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Long interspersed nuclear element-1 hypomethylation in cancer: biology and clinical applications
Clinical Epigenetics volume 2, pages315–330(2011)
Epigenetic changes in long interspersed nuclear element-1s (LINE-1s or L1s) occur early during the process of carcinogenesis. A lower methylation level (hypomethylation) of LINE-1 is common in most cancers, and the methylation level is further decreased in more advanced cancers. Consequently, several previous studies have suggested the use of LINE-1 hypomethylation levels in cancer screening, risk assessment, tumor staging, and prognostic prediction. Epigenomic changes are complex, and global hypomethylation influences LINE-1s in a generalized fashion. However, the methylation levels of some loci are dependent on their locations. The consequences of LINE-1 hypomethylation are genomic instability and alteration of gene expression. There are several mechanisms that promote both of these consequences in cis. Therefore, the methylation levels of different sets of LINE-1s may represent certain phenotypes. Furthermore, the methylation levels of specific sets of LINE-1s may indicate carcinogenesis-dependent hypomethylation. LINE-1 methylation pattern analysis can classify LINE-1s into one of three classes based on the number of methylated CpG dinucleotides. These classes include hypermethylation, partial methylation, and hypomethylation. The number of partial and hypermethylated loci, but not hypomethylated LINE-1s, is different among normal cell types. Consequently, the number of hypomethylated loci is a more promising marker than methylation level in the detection of cancer DNA. Further genome-wide studies to measure the methylation level of each LINE-1 locus may improve PCR-based methylation analysis to allow for a more specific and sensitive detection of cancer DNA or for an analysis of certain cancer phenotypes.
Because of the retrotransposition events that have occurred during evolution, the human genome contains more than 500,000 long interspersed nuclear element-1 (LINE-1 or L1) copies (Lander et al. 2001). Most LINE-1s are truncated. More than 10,000 LINE-1s are longer than 4.5 kb and consist of a 5′ untranslated region (UTR), two open reading frames, and a 3′ UTR containing a polyadenylation signal (Penzkofer et al. 2005). The DNA methylation levels of LINE-1 5′ UTRs in cancer have been extensively evaluated for potential use as an epigenomic marker for cancer (Chalitchagorn et al. 2004). The mean level of LINE-1 methylation in most cancer types is lower than in normal cells (Table 1). The degree of LINE-1 hypomethylation increases in more advanced cancers (Table 2 and Electronic supplementary material (ESM) Table 1). The methylation of other interspersed repetitive sequences (IRSs), such as Alu elements and human endogenous retrovirus (HERV) sequences, has been evaluated to a lesser extent (Tables 1 and 2 and ESM Table 1). LINE-1 and other IRS methylation levels have the potential to be used as universal tumor markers for the detection of cancer DNA and to predict prognosis (Watanabe and Maekawa 2010).
LINE-1s have often been referred to as parasitic or junk DNA sequences. However, many LINE-1s play a role in gene regulation, and this control is regulated by the 5′ UTR methylation level (Aporntewan et al. 2011). As a result, changes in the methylation status of different sets of LINE-1 loci may lead to different cellular phenotypes (Phokaew et al. 2008; Aporntewan et al. 2011). These differences may be an underlying reason why LINE-1 methylation levels in normal cells show so much variation (Chalitchagorn et al. 2004). Lower methylation levels can also be found in many non-malignant conditions. Current PCR-based techniques were designed to measure LINE-1 methylation level and cannot distinguish between malignant- and non-malignant-associated LINE-1 hypomethylation (Xiong and Laird 1997; Laird 2010; Weisenberger et al. 2005; Yang et al. 2004). Therefore, a technique that measures not only the level but also the pattern of LINE-1 methylation should improve detection specificity and sensitivity and broaden the applications of this tumor marker. The topics of this review therefore include the following: (1) an up-to-date review of studies on LINE-1 and other IRS methylation levels in cancer; (2) the characteristics of LINE-1 hypomethylation in cancer; (3) the locus-dependent roles of LINE-1 hypomethylation in cancer development; and (4) the improvement in cancer DNA identification by LINE-1 methylation classification.
LINE-1 and other IRS methylation levels in cancer
The methylation levels of LINE-1s, Alu elements, and some types of HERVs have been studied (Table 1). LINE-1 is the IRS element that is most frequently studied, and its hypomethylation has been found in many cancers. In a few cancer types, including cancer of the kidney, thyroid, and lymph nodes; acute promyelocytic leukemia; malignant peripheral nerve sheath tumor; and parathyroid adenoma, LINE-1 hypomethylation had not been found (Table 1). LINE-1 hypomethylation is also found in premalignant lesions of the cervix (Shuangshoti et al. 2007), extrahepatic bile duct (Kim et al. 2009a), and stomach (Park et al. 2009). Unexpectedly, LINE-1 hypermethylation was observed in some lesions that possess a high potential for malignant transformation, including lesions associated with myelodysplastic syndrome (Romermann et al. 2007) and liver cirrhosis (Takai et al. 2000). Interestingly, LINE-1 hypermethylation is found in partial hydatidiform moles, whereas LINE-1 hypomethylation is seen in triploid diandric embryos. Both lesions originate from dispermic fertilization of an oocyte, suggesting that LINE-1 hypermethylation in moles is directly linked to the neoplastic process and is not a consequence of growth control (Perrin et al. 2007).
As shown in Table 2 and ESM Table 1, LINE-1 hypomethylation is associated with advanced tumor stage, higher histological grade, and poor prognosis. LINE-1 hypomethylation increases with tumor size (Tangkijvanich et al. 2007) and with higher tumor stage (Florl et al. 1999; Kindich et al. 2006; Pattamadilok et al. 2008; Lee et al. 2009; Baba et al. 2010). With increasing histological grade, according to multistep carcinogenesis, LINE-1 hypomethylation levels are increased in many cancer types (Florl et al. 1999; Shuangshoti et al. 2007; Cho et al. 2007; Park et al. 2009; Iramaneerat et al. 2011; Pattamadilok et al. 2008). Furthermore, LINE-1 hypomethylation is correlated with chromosomal aberrations (Schulz et al. 2002; Cho et al. 2007; Choi et al. 2007; Ogino et al. 2008a; Bollati et al. 2009), the hypermethylation of tumor suppressor genes (Choi et al. 2007; Kim et al. 2009a), mutations of tumor suppressor genes (Iacopetta et al. 2007; Kim et al. 2009a), the alternate transcription of oncogenes (Wolff et al. 2010), and the deregulation of cancer genes (Woloszynska-Read et al. 2008). Therefore, LINE-1 hypomethylation is associated with malignant phenotypes in human cells, deregulating gene expression and accelerating DNA rearrangement. Interestingly, the LINE-1 hypomethylation level is inversely associated with microsatellite instability (Estecio et al. 2007; Iacopetta et al. 2007; Ogino et al. 2008a; Goel et al. 2010; Kawakami et al. 2011). This finding may indicate that microsatellite instability and LINE-1 hypomethylation are characteristics of different genomic instability mechanisms.
From a clinical point of view, LINE-1 hypomethylation is associated with tumor metastasis (Schulz et al. 2002; Choi et al. 2007), the recurrence rate (Formeister et al. 2010), and the mortality rate (Ogino et al. 2008b; Ahn et al. 2011). LINE-1 hypomethylation has been reported to be a prognostic marker in several types of cancer including the stage IA subgroup of non-small cell lung cancer (Saito et al. 2010), ovary (Pattamadilok et al. 2008), and colon (Ogino et al. 2008b; Baba et al. 2010). LINE-1 hypomethylation has been proposed to be used as a screening tool for cancer detection. LINE-1 hypomethylation is observed in blood leukocyte DNA (Hsiung et al. 2007; Wilhelm et al. 2010), serum (Chalitchagorn et al. 2004; Tangkijvanich et al. 2007), and oral rinse samples (Subbalekha et al. 2009). Moreover, LINE-1 hypomethylation has also been demonstrated to be a surrogate marker for predicting tumor treatment response and prognosis (Aparicio et al. 2009; Sonpavde et al. 2009; Bernstein et al. 2010; Fang et al. 2010; Kawakami et al. 2011).
Alu elements and HERV genomes have been studied less frequently (Table 1). Hypomethylation of Alu sequences was reported in nine cancers, whereas hypomethylation of HERV-K and HERV-W genomes was found in urothelial cancer (Florl et al. 1999) and ovarian cancer (Menendez et al. 2004), respectively. All of the Alu- and HERV-hypomethylated cancers also possess LINE-1 hypomethylation. Certain cancer phenotypes are associated with the methylation levels of certain IRS types. For example, HERV-K, but not LINE-1 and HERV-E, methylation levels are associated with poor prognosis and platinum resistance of ovarian clear cell carcinoma (Iramaneerat et al. 2011).
Characteristics of LINE-1 and global hypomethylation in cancer
Transgenic mice with hereditary defects in DNA methyltransferase show increased risk of developing cancer (Gaudet et al. 2003). Therefore, global hypomethylation may be one of the mechanisms that promote carcinogenesis and is unlikely to be just a consequence of cancer development. However, lower genome-wide methylation levels have also been found in many conditions, such as embryogenesis (Migeon et al. 1991; Kremenskoy et al. 2003), aging (Lutz et al. 1972; Gonzalo 2010), congenital malformation (Wang et al. 2010), exposure to certain environments (Bollati et al. 2007), nutrition (Brunaud et al. 2003), and autoimmune diseases (Richardson et al. 1990). There is no report of increased cancer development risk in individuals with some of these conditions. Therefore, it is reasonable to hypothesize that the genomic distribution of IRS methylation levels is different in global hypomethylation-related conditions. Interestingly, in some conditions, the loss of genome-wide methylation is IRS type-specific. For example, hypomethylation of Alu elements and HERV-K, but not LINE-1, was found in aging cells (Jintaridth and Mutirangura 2011). However, LINE-1 hypomethylation has been demonstrated in many other conditions (Schulz et al. 2006). Because LINE-1 methylation levels can regulate host gene expression in cis (Aporntewan et al. 2011), it is reasonable to hypothesize that the reduction in LINE-1 methylation is the result of epigenomic heterogeneity. A simpler explanation is that even though two different cells possess the same number of LINE-1 loci and methylation levels, each LINE-1 locus may have a different level of LINE-1 methylation in these cells (Phokaew et al. 2008). Therefore, LINE-1 hypomethylation is a cancer biomarker that may be a diagnostic tool for many cancer types. However, LINE-1 hypomethylation is not specific to cancer. The inclusion of information regarding the genomic LINE-1 methylation distribution pattern should therefore be a promising way to improve and widen the applications of LINE-1 methylation as a tumor marker (Pobsook et al. 2011).
Although LINE-1 methylation levels are variable in both cancer and normal cells, the mechanisms that alter methylation levels may be different. Normal cells possess several patterns of LINE-1 methylation levels. The levels of some cell types are precise and limited to within a specific range. In other cases, such as in the esophagus and thyroid, the ranges are expanded (Chalitchagorn et al. 2004). Similar patterns can be observed when the methylation status of each LINE-1 locus is observed (Phokaew et al. 2008). Different loci possess different methylation levels. Some are limited in range and others have wider ranges. Levels of LINE-1 locus methylation between different cell types are usually different, but each locus reveals similar patterns regarding the range of methylation levels (Phokaew et al. 2008).
Comparison of methylation levels between LINE-1 loci in normal cells showed no significant correlation. This result suggests that the methylation level is locus-dependent (Fig. 1; Phokaew et al. 2008). In contrast, significant associations of methylation levels between LINE-1 loci were frequently found in cancer. Therefore, the mechanism causing LINE-1 hypomethylation in cancer occurs generally and in a genome-wide manner (Fig. 1; Phokaew et al. 2008). However, this mechanism may be biased toward some IRS sequences. Using microarray analysis, Szpakowski et al. (2009) reported that primate-specific LINE-1 elements and most of the younger, primate-specific retroelements were preferentially hypomethylated in samples of squamous cell carcinoma of the head and neck in comparison to non-tumor adjacent tissue and normal controls. The association of the methylation level between two LINE-1 loci was found to be highest if they were located in the same gene (Phokaew et al. 2008). Therefore, in addition to evolutionarily derived classifications, LINE-1 hypomethylation in cancer can be influenced by genomic location.
LINE-1 methylation regulates gene expression in cis
The notion that LINE-1 is methylated to prevent the process of retrotransposition should be reevaluated. First, in the human genome, less than 100 LINE-1s are retrotransposition competent, and only a few LINE-1s have been shown to be responsible for retrotransposition events during human evolution (Sassaman et al. 1997). Although a recent study showed that LINE-1 retrotransposition may be common (Lupski 2010; Beck et al. 2010), this evidence fails to explain the methylation of the vast majority of retrotransposition-incompetent LINE-1s. The human genome possesses thousands of 5′ UTR-containing LINE-1s, and most of them are methylated to a certain degree (Chalitchagorn et al. 2004). It is unlikely that this methylation provides a selective advantage to the cells by preventing retrotransposition. The significant differences in LINE-1 methylation levels between loci or cell types suggests that LINE-1 methylation may be important to maintain normal cellular function and that this function may be altered by the global hypomethylation process that occurs in cancer.
The location-dependent LINE-1 methylation pattern in normal cells suggests a role for epigenetic regulation. Currently, there are at least two reported mechanisms for how LINE-1 methylation regulates gene expression in cis. Both mechanisms are dependent on the transcriptional activity of the LINE-1 promoter. Moreover, similar to other promoters, the LINE-1 5′ UTR promoter is controlled by DNA methylation, and the transcription activity of a LINE-1 element is directly correlated with its hypomethylation level (Aporntewan et al. 2011). The first mechanism is that LINE-1-mediated control of gene expression is through the production of unique RNA sequences (Fig. 2). The other mechanism is that intragenic LINE-1 RNAs repress host gene expression via the nuclear RNA-induced silencing complex (RISC; Fig. 3).
There are two ways for the LINE-1 promoter to produce unique RNA sequences (Fig. 2). The 5′ UTR of LINE-1 is a promoter that transcribes in both the forward and reverse directions (Matlik et al. 2006; Weber et al. 2010; Speek 2001; Wolff et al. 2010; Rangwala et al. 2009). If the transcription is in the forward orientation, then the promoter produces LINE-1 RNA. However, the poly-A addition signal of LINE-1 does not always function. Consequently, many LINE-1 transcripts can continue beyond the end of the LINE-1 sequence, therefore resulting in 3′ transduction (Moran et al. 1999; Rangwala et al. 2009). These transduction sequences are unique RNA sequences generated by the LINE-1 promoter. On the other hand, LINE-1 5′ transduction that occurs by reverse transcription will also produce unique RNA sequences. A large number of these transduction sequences have been reported (Rangwala et al. 2009); however, there are currently only two examples that prove that these sequences are increased by LINE-1 hypomethylation (Weber et al. 2010; Wolff et al. 2010; Aporntewan et al. 2011).
Intragenic LINE-1 regulation of host gene expression was revealed by the finding that in vitro insertion of a full-length LINE-1 disrupted host gene expression (Han et al. 2004). In vivo, this gene regulation is tuned by LINE-1 methylation levels (Aporntewan et al. 2011). When LINE-1 methylation levels were reduced by chemical treatment or by carcinogenesis, a significant number of genes containing LINE-1s were repressed (Fig. 3a–c). The degree of this repression was inversely correlated with the intragenic LINE-1 methylation level. The role of LINE-1 methylation is to prevent the formation of a pre-mRNA–LINE-1–RNA complex. If the complex is formed, then the RISC protein AGO2 will bind and prevent mRNA production (Fig. 3; Aporntewan et al. 2011).
Comparative sequence analysis between intragenic and intergenic LINE-1s showed multiple conserved nucleotides in intragenic LINE-1s that are crucial for maintaining LINE-1 transcription and methylation (Aporntewan et al. 2011). Moreover, many LINE-1s are excluded from genomic regions containing housekeeping genes (Eller et al. 2007; Graham and Boissinot 2006). Therefore, locations of LINE-1s yield a selective advantage for human evolution. It is important to note that the diploid human genome contains an extensive amount of structural variation due to retrotransposition events (Huang et al. 2010; Ewing and Kazazian 2011). Consequently, variation in the expression of many genes may be due to the distinctive locations of heritable LINE-1s, and similar to other DNA polymorphisms, some LINE-1 insertions are polymorphisms that lead to certain disease-related phenotypes. LINE-1 hypomethylation may also control gene expression in trans. In some cancer cells, inhibition of LINE-1 reverse transcriptase can alter the expression of many genes (Carlini et al. 2010).
LINE-1 hypomethylation and genomic instability in cancer
In addition to a number of association studies (Ji et al. 1997; Lu and Randerath 1984; Daskalos et al. 2009), the high risk of chromosomal abnormalities in individuals with hereditary mutations in DNA methyltransferase genes indicates that global hypomethylation promotes genomic instability (Hansen et al. 1999; Eden et al. 2003). However, the underlying mechanisms of how DNA methylation maintains genomic integrity are not yet known. Current reports suggest that LINE-1 hypomethylation leads to several events that promote genomic instability, including retrotransposition, endogenous DNA double-strand break (EDSB) repair, and the dysregulation of DNA repair genes.
The process of LINE-1 retrotransposition includes RNA transcription, protein translation, DNA restriction, reverse transcription, and integration (Moran 1999). This retrotransposition usually produces large DNA rearrangements (Huang et al. 2010; Gilbert et al. 2002). Recently, an advanced LINE-1 junction sequencing technique showed that somatic L1 insertions occur at high frequency in human lung cancer genomes (Iskow et al. 2010). Therefore, LINE-1 hypomethylation in cancer may increase the retrotransposition activity of some LINE-1s and consequently cause a faster rate of DNA rearrangement. However, many DNA rearrangements occur in cancer cells that are not LINE-1 retrotransposition events. Therefore, LINE-1 retrotransposition contributes to only a small proportion of mutations in cancer. Moreover, there are only a few reports that retrotransposition events can produce clonal expansion mutations (Miki et al. 1992). Finally, the loss of the methylation of non-retrotransposable repeats, such as satellite DNA, also promotes chromosome translocation (Maraschio et al. 1988; Ji et al. 1997). Therefore, LINE-1 retrotransposition may not be the major mechanism causing somatic mutation in cancer by global hypomethylation.
The second mechanism is the differential repair of methylated and unmethylated replication-independent EDSBs (RIND-EDSBs; Kongruttanachok et al. 2010). RIND-EDSBs are different from replication-dependent EDSBs and environmental- or radiation-induced DSBs. Replication-dependent EDSBs and radiation-induced DSBs, if unrepaired, lead to cell death. In contrast, RIND-EDSBs are ubiquitously present in all cells and always involve hypermethylation (Pornthanakasem et al. 2008). This occurrence indicates a time lag between methylated RIND-EDSB production and repair (Kongruttanachok et al. 2010). RIND-EDSBs can be produced within both methylated and unmethylated genomes. Methylated RIND-EDSBs are selectively repaired by the more precise ataxia telangiectasia mutated (ATM)-dependent non-homologous end joining repair process (Kongruttanachok et al. 2010). Therefore, the RIND-EDSB repair process of hypomethylated genomes is faster and more error-prone. Because the LINE-1 methylation levels of each locus are distinct, the mutation rate caused by RIND-EDSB repair errors is dependent on the methylation status of the genome near the EDSBs. Currently, there are only two reports focused on RIND-EDSBs (Pornthanakasem et al. 2008; Kongruttanachok et al. 2010). Further studies are needed to explore the causes and roles of RIND-EDSBs and to determine how genomic hypomethylation promotes instability.
A third possible mechanism is that LINE-1 hypomethylation down-regulates DNA repair genes. One of these genes is PPP2R2B, which contains intragenic LINE-1s. In cancer, these LINE-1s are frequently hypomethylated and PPP2R2B is frequently down-regulated (Aporntewan et al. 2011). One of the functions of PPP2R2B is to increase nuclear ATM protein (Suyarnsestakorn et al. 2010). ATM is a serine/threonine protein kinase that is important in the activation of the DNA damage checkpoint, leading to cell cycle arrest, DNA repair, or apoptosis (Mavrou et al. 2008). A lack of ATM promotes genomic instability (Kim et al. 2002). Therefore, LINE-1 hypomethylation may indirectly promote genomic instability by interfering with ATM function.
LINE-1 methylation patterns in normal and cancer cells
It is commonly assumed that LINE-1 elements in normal cells are completely methylated. Combined bisulfite restriction analysis or COBRA, deep sequencing, and microarray analysis demonstrated that the genomic distribution of the methylation of LINE-1s and other IRS loci is not homogenous (Phokaew et al. 2008; Xie et al. 2009, 2011; Szpakowski et al. 2009). The methylation levels of LINE-1 loci can be divided into three groups: hypermethylated, partially methylated, and hypomethylated (Pobsook et al. 2011). Classification is based on the number of methylated and unmethylated CpG dinucleotides (Fig. 1). In normal cells, the majorities of LINE-1 loci are hypermethylated or partially methylated. Few LINE-1 loci are hypomethylated. Comparisons between normal white blood cells and normal oral epithelium showed that even though LINE-1 methylation levels are different, the number of hypomethylated loci was not distinguishable between the two normal tissues (Fig. 4). Therefore, the differences in methylation levels between normal cell types are primarily influenced by the number of hypermethylated and partially methylated loci. In cancer cells, the methylation of a majority of LINE-1 loci is decreased, with some loci remaining unchanged and a few being increased when compared with normal cells. Thus, the number of hypomethylated loci is increased in cancer cells (Fig. 4).
A recent report showed distinctive characteristics of LINE-1 partial methylation that was dependent on malignant transformation (Pobsook et al. 2011). In normal cells, the number of partially methylated LINE-1 loci in each sample was directly correlated with the number of hypomethylated loci, but was inversely associated with the number of hypermethylated loci. This result suggested that a dynamic form of LINE-1 epigenetic modification, between partial methylation and hypermethylation, is present in normal cells. Because hypomethylated LINE-1s were not distinguishable between different types of normal cells, the dynamic between the partially methylated and hypermethylated forms may be the cause of the variation in LINE-1 methylation levels between normal cell types. Moreover, the more partially methylated loci may represent the lower LINE-1 methylation level. In contrast, in the cancer genome, the number of partially methylated LINE-1s was directly correlated with the number of hypermethylated LINE-1s. Therefore, in striking contrast to the normal genome, partially methylated LINE-1 loci represent a subset of methylated LINE-1s in cancer cells (Pobsook et al. 2011). Current PCR-based techniques, by real-time quantitative PCR, COBRA, and pyrosequencing, determine LINE-1 hypomethylation levels by combining all unmethylated CpG nucleotides from both partially methylated or hypomethylated loci (Xiong and Laird 1997; Laird 2010; Weisenberger et al. 2005; Yang et al. 2004). Therefore, the sensitivity in distinguishing cancer DNA is low. Pobsook et al. (2011) also showed that excluding partial methylation loci from the count of hypomethylated LINE-1 loci improved the sensitivity and specificity of cancer DNA detection.
From biology to clinical application and future direction of LINE-1 hypomethylation in cancer
Understanding how LINE-1 methylation levels change during multistep carcinogenesis has implications for diagnostic applications. Several LINE-1 and other IRS methylation studies have shown that global hypomethylation is a common epigenetic change in cancer (Table 1). Moreover, this process is directly correlated with cancer progression. Therefore, lower LINE-1 methylation levels have been shown to be associated with higher cancer stages and may also be a promising marker for the prognostic prediction of many cancers (Table 2 and ESM Table 1). Global methylation changes initiate early, and the genome becomes progressively hypomethylated during the process of multistep carcinogenesis. Therefore, LINE-1 and other IRS hypomethylation levels are candidate tumor markers for cancer (Table 2 and ESM Table 1).
There is a technical advantage to using PCR-based assays to measure IRS methylation levels. Multiple copies of IRSs are present in the genome; therefore, this detection method is highly sensitive even in poor-quality clinical DNA samples. These clinical samples include paraffin-embedded sections, plasma, and other fluid or washes, such as oral rinses (Chalitchagorn et al. 2004; Tangkijvanich et al. 2007; Aparicio et al. 2009; Subbalekha et al. 2009) (Vaissiere et al. 2009). LINE-1 hypomethylation was also detected in the white blood cells of cancer patients (Hsiung et al. 2007; Wilhelm et al. 2010). The source of the hypomethylated cells in cancer patients still needs to be identified to determine whether these cells are from cancer cells or from normal cells with systemic hypomethylated LINE-1s. Nevertheless, this evidence suggests that LINE-1 methylation is a promising marker in cancer risk prediction.
Cells must have a correct amount of LINE-1 methylation to maintain their physiological functions (Aporntewan et al. 2011). Consequently, there is a wide range of LINE-1 methylation levels found in normal cells, depending on cell type (Chalitchagorn et al. 2004). This methylation range leads to low specificity when using LINE-1 hypomethylation as a cancer screening marker. The ability to distinguish between normal and tumor DNA is low, particularly because clinical samples, including plasma, mouth washes, or Papanicolaou smears, are routinely contaminated with DNA from several normal cell types. LINE-1 methylation pattern analysis demonstrated unprecedented characteristics of LINE-1 partial methylation in normal cells and in the cancer global hypomethylation process (Pobsook et al. 2011). The interchangeable pattern between LINE-1 hypermethylation and partial methylation is a mechanism that may result in different LINE-1 methylation levels in normal cells (Pobsook et al. 2011). In cancer, global hypomethylation is observed because of the loss of methylation of previously hypermethylated and partial methylated loci. Most PCR-based LINE-1 methylation measurement techniques cannot differentiate unmethylated CpG dinucleotides of partially methylated LINE-1s from unmethylated LINE-1s. There was a recent report using COBRA to classify LINE-1s into the three classes. This report showed that the number of unmethylated LINE-1 loci was a more sensitive and specific marker than LINE-1 methylation level to detect cancer DNA in mouthwash samples (Pobsook et al. 2011). It may be interesting to compare the number of unmethylated LINE-1 loci with LINE-1 methylation levels in other clinical samples. Moreover, it may be worth exploring whether changes in partially methylated LINE-1 loci can be observed in, and are able to predict, malignant transformation in pathological lesions in the very early stages of carcinogenesis or tissues in patients at risk of developing cancer.
Although the methylation of a majority of LINE-1 loci is reduced in cancer, some loci are unchanged. Currently, there are several advanced genomic techniques, including deep sequencing (Xie et al. 2009; Xie et al. 2011) and custom-made microarrays (Szpakowski et al. 2009), that are capable of measuring the methylation level of each LINE-1 or IRS locus. These approaches identified certain classes of LINE-1s and IRSs that more frequently show loss of methylation in cancer. Improved deep sequencing techniques will be able to determine the proportions of the three LINE-1 methylation classes at each LINE-1 locus. It is important to reevaluate the clinical significance of LINE-1 methylation by these advanced techniques. These methods should help define the relevant LINE-1 locations, sequences, and methylation patterns that are specific to carcinogenesis. Moreover, some intragenic LINE-1 loci are methylated cis-regulatory elements of their host genes (Aporntewan et al. 2011). Altered expression of these genes may lead to certain cellular phenotypes and clinical presentations. Genome-wide arrays or deep sequencing may be used to design promising new sets of methylated LINE-1 PCR-based techniques specifically aimed for the classification of the epigenome of the tumor phenotype.
Interestingly, some pathological lesions with increased potential for malignant transformation, such as myelodysplastic syndrome lesions, liver cirrhosis, and partial hydatidiform moles, possess LINE-1 hypermethylation (Takai et al. 2000; Romermann et al. 2007; Perrin et al. 2007). Further descriptive studies of other lesions, genomic distributions, and methylation patterns will clarify in detail whether this epigenetic process occurs during the early steps of LINE-1 hypomethylation in cancer. It is important to note that genome-wide hypomethylation in cancer can result in hypermethylated LINE-1s at some loci (Fig. 4; Phokaew et al. 2008). If LINE-1 hypermethylation and hypomethylation are present at the same loci in premalignant tissues and cancer, this finding would be a breakthrough by showing that epigenomic changes precede genetic changes during carcinogenesis. Detailed molecular biological approaches to explain how LINE-1 methylation fluctuates from hypermethylation to hypomethylation will be important to understand the development of global hypomethylation in cancer.
Finally, global hypomethylation mechanisms may be crucial for future cancer prevention and treatment. Genome-wide hypomethylation is common, occurs at an earlier stage of carcinogenesis, and is still an active process in most cancers (Tables 1 and 2 and ESM Table 1). Global hypomethylation is an epigenomic process that leads to cellular phenotypic changes. LINE-1 hypomethylation in cancer alters the expression of a large number of genes. Therefore, this epigenomic alteration should be an important target for future cancer prevention strategies. Moreover, unlike mutation, hypomethylation is reversible. Therefore, global hypomethylation in cancer is a candidate for new cancer treatments in the future.
Ahn JB, Chung WB, Maeda O, Shin SJ, Kim HS, Chung HC, Kim NK, Issa JP (2011) DNA methylation predicts recurrence from resected stage III proximal colon cancer. Cancer. doi:10.1002/cncr.25737
Alves G, Tatro A, Fanning T (1996) Differential methylation of human LINE-1 retrotransposons in malignant cells. Gene 176(1–2):39–44
An B, Kondo Y, Okamoto Y, Shinjo K, Kanemitsu Y, Komori K, Hirai T, Sawaki A, Tajika M, Nakamura T, Yamao K, Yatabe Y, Fujii M, Murakami H, Osada H, Tani T, Matsuo K, Shen L, Issa JP, Sekido Y (2010) Characteristic methylation profile in CpG island methylator phenotype-negative distal colorectal cancers. Int J Cancer 127(9):2095–2105. doi:10.1002/ijc.25225
Aparicio A, North B, Barske L, Wang X, Bollati V, Weisenberger D, Yoo C, Tannir N, Horne E, Groshen S, Jones P, Yang A, Issa JP (2009) LINE-1 methylation in plasma DNA as a biomarker of activity of DNA methylation inhibitors in patients with solid tumors. Epigenetics 4(3):176–184. doi:8694
Aporntewan C, Phokaew C, Piriyapongsa J, Ngamphiw C, Ittiwut C, Tongsima S, Mutirangura A (2011) Hypomethylation of intragenic LINE-1 represses transcription in cancer cells through AGO2. PLoS ONE 6(3):e17934
Baba Y, Huttenhower C, Nosho K, Tanaka N, Shima K, Hazra A, Schernhammer ES, Hunter DJ, Giovannucci EL, Fuchs CS, Ogino S (2010) Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer 9:125. doi:10.1186/1476-4598-9-125
Beck CR, Collier P, Macfarlane C, Malig M, Kidd JM, Eichler EE, Badge RM, Moran JV (2010) LINE-1 retrotransposition activity in human genomes. Cell 141(7):1159–1170. doi:10.1016/j.cell.2010.05.021
Bernstein I, Byun HM, Mohrbacher A, Douer D, Gorospe G 3rd, Hergesheimer J, Groshen S, O’Connell C, Yang AS (2010) A phase I biological study of azacitidine (VidazaTM) to determine the optimal dose to inhibit DNA methylation. Epigenetics 5(8):750–757. doi:10.4161/epi.5.8.13105
Bollati V, Baccarelli A, Hou L, Bonzini M, Fustinoni S, Cavallo D, Byun HM, Jiang J, Marinelli B, Pesatori AC, Bertazzi PA, Yang AS (2007) Changes in DNA methylation patterns in subjects exposed to low-dose benzene. Cancer Res 67(3):876–880. doi:10.1158/0008-5472.CAN-06-2995
Bollati V, Fabris S, Pegoraro V, Ronchetti D, Mosca L, Deliliers GL, Motta V, Bertazzi PA, Baccarelli A, Neri A (2009) Differential repetitive DNA methylation in multiple myeloma molecular subgroups. Carcinogenesis 30(8):1330–1335. doi:10.1093/carcin/bgp149
Brunaud L, Alberto JM, Ayav A, Gerard P, Namour F, Antunes L, Braun M, Bronowicki JP, Bresler L, Gueant JL (2003) Effects of vitamin B12 and folate deficiencies on DNA methylation and carcinogenesis in rat liver. Clin Chem Lab Med 41(8):1012–1019. doi:10.1515/CCLM.2003.155
Carlini F, Ridolfi B, Molinari A, Parisi C, Bozzuto G, Toccacieli L, Formisano G, De Orsi D, Paradisi S, Grober OM, Ravo M, Weisz A, Arcieri R, Vella S, Gaudi S (2010) The reverse transcription inhibitor abacavir shows anticancer activity in prostate cancer cell lines. PLoS ONE 5(12):e14221. doi:10.1371/journal.pone.0014221
Chalitchagorn K, Shuangshoti S, Hourpai N, Kongruttanachok N, Tangkijvanich P, Thong-ngam D, Voravud N, Sriuranpong V, Mutirangura A (2004) Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis. Oncogene 23(54):8841–8846. doi:10.1038/sj.onc.1208137
Cho NY, Kim BH, Choi M, Yoo EJ, Moon KC, Cho YM, Kim D, Kang GH (2007) Hypermethylation of CpG island loci and hypomethylation of LINE-1 and Alu repeats in prostate adenocarcinoma and their relationship to clinicopathological features. J Pathol 211(3):269–277. doi:10.1002/path.2106
Cho NY, Kim JH, Moon KC, Kang GH (2009) Genomic hypomethylation and CpG island hypermethylation in prostatic intraepithelial neoplasm. Virchows Arch 454(1):17–23. doi:10.1007/s00428-008-0706-6
Cho YH, Yazici H, Wu HC, Terry MB, Gonzalez K, Qu M, Dalay N, Santella RM (2010) Aberrant promoter hypermethylation and genomic hypomethylation in tumor, adjacent normal tissues and blood from breast cancer patients. Anticancer Res 30(7):2489–2496. doi:30/7/2489
Choi IS, Estecio MR, Nagano Y, Kimdo H, White JA, Yao JC, Issa JP, Rashid A (2007) Hypomethylation of LINE-1 and Alu in well-differentiated neuroendocrine tumors (pancreatic endocrine tumors and carcinoid tumors). Mod Pathol 20(7):802–810. doi:10.1038/modpathol.3800825
Choi SH, Worswick S, Byun HM, Shear T, Soussa JC, Wolff EM, Douer D, Garcia-Manero G, Liang G, Yang AS (2009) Changes in DNA methylation of tandem DNA repeats are different from interspersed repeats in cancer. Int J Cancer 125(3):723–729. doi:10.1002/ijc.24384
Dammann RH, Kirsch S, Schagdarsurengin U, Dansranjavin T, Gradhand E, Schmitt WD, Hauptmann S (2010) Frequent aberrant methylation of the imprinted IGF2/H19 locus and LINE1 hypomethylation in ovarian carcinoma. Int J Oncol 36(1):171–179
Daskalos A, Nikolaidis G, Xinarianos G, Savvari P, Cassidy A, Zakopoulou R, Kotsinas A, Gorgoulis V, Field JK, Liloglou T (2009) Hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer. Int J Cancer 124(1):81–87. doi:10.1002/ijc.23849
Deng G, Nguyen A, Tanaka H, Matsuzaki K, Bell I, Mehta KR, Terdiman JP, Waldman FM, Kakar S, Gum J, Crawley S, Sleisenger MH, Kim YS (2006) Regional hypermethylation and global hypomethylation are associated with altered chromatin conformation and histone acetylation in colorectal cancer. Int J Cancer 118(12):2999–3005. doi:10.1002/ijc.21740
Eden A, Gaudet F, Waghmare A, Jaenisch R (2003) Chromosomal instability and tumors promoted by DNA hypomethylation. Science 300(5618):455. doi:10.1126/science.1083557
Eller CD, Regelson M, Merriman B, Nelson S, Horvath S, Marahrens Y (2007) Repetitive sequence environment distinguishes housekeeping genes. Gene 390(1–2):153–165. doi:10.1016/j.gene.2006.09.018
Estecio MR, Gharibyan V, Shen L, Ibrahim AE, Doshi K, He R, Jelinek J, Yang AS, Yan PS, Huang TH, Tajara EH, Issa JP (2007) LINE-1 hypomethylation in cancer is highly variable and inversely correlated with microsatellite instability. PLoS ONE 2(5):e399. doi:10.1371/journal.pone.0000399
Ewing AD, Kazazian HH, Jr. (2011) Whole-genome resequencing allows detection of many rare LINE-1 insertion alleles in humans. Genome Res. doi:10.1101/gr.114777.110
Fabris S, Bollati V, Agnelli L, Morabito F, Motta V, Cutrona G, Matis S, Recchia AG, Gigliotti V, Gentile M, Deliliers GL, Bertazzi PA, Ferrarini M, Neri A, Baccarelli A (2011) Biological and clinical relevance of quantitative global methylation of repetitive DNA sequences in chronic lymphocytic leukemia. Epigenetics 6(2):188–194. doi:13528
Fang F, Balch C, Schilder J, Breen T, Zhang S, Shen C, Li L, Kulesavage C, Snyder AJ, Nephew KP, Matei DE (2010) A phase 1 and pharmacodynamic study of decitabine in combination with carboplatin in patients with recurrent, platinum-resistant, epithelial ovarian cancer. Cancer 116(17):4043–4053. doi:10.1002/cncr.25204
Feber A, Wilson GA, Zhang L, Presneau N, Idowu B, Down TA, Rakyan VK, Noon LA, Lloyd AC, Stupka E, Schiza V, Teschendorff AE, Schroth GP, Flanagan AM, Beck S (2011) Comparative methylome analysis of benign and malignant peripheral nerve sheath tumors. Genome Res 21:515–524. doi:10.1101/gr.109678.110
Florl AR, Lower R, Schmitz-Drager BJ, Schulz WA (1999) DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas. Br J Cancer 80(9):1312–1321. doi:10.1038/sj.bjc.6690524
Florl AR, Steinhoff C, Muller M, Seifert HH, Hader C, Engers R, Ackermann R, Schulz WA (2004) Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-1 hypomethylation. Br J Cancer 91(5):985–994. doi:10.1038/sj.bjc.6602030
Formeister EJ, Tsuchiya M, Fujii H, Shpyleva S, Pogribny IP, Rusyn I (2010) Comparative analysis of promoter methylation and gene expression endpoints between tumorous and non-tumorous tissues from HCV-positive patients with hepatocellular carcinoma. Mutat Res 692(1–2):26–33. doi:10.1016/j.mrfmmm.2010.07.013
Furniss CS, Marsit CJ, Houseman EA, Eddy K, Kelsey KT (2008) Line region hypomethylation is associated with lifestyle and differs by human papillomavirus status in head and neck squamous cell carcinomas. Cancer Epidemiol Biomark Prev 17(4):966–971. doi:10.1158/1055-9965.EPI-07-2775
Gaudet F, Hodgson JG, Eden A, Jackson-Grusby L, Dausman J, Gray JW, Leonhardt H, Jaenisch R (2003) Induction of tumors in mice by genomic hypomethylation. Science 300(5618):489–492. doi:10.1126/science.1083558
Geli J, Kiss N, Karimi M, Lee JJ, Backdahl M, Ekstrom TJ, Larsson C (2008) Global and regional CpG methylation in pheochromocytomas and abdominal paragangliomas: association to malignant behavior. Clin Cancer Res 14(9):2551–2559. doi:10.1158/1078-0432.CCR-07-1867
Gilbert N, Lutz-Prigge S, Moran JV (2002) Genomic deletions created upon LINE-1 retrotransposition. Cell 110(3):315–325. doi:S0092867402008280
Goel A, Xicola RM, Nguyen TP, Doyle BJ, Sohn VR, Bandipalliam P, Rozek LS, Reyes J, Cordero C, Balaguer F, Castells A, Jover R, Andreu M, Syngal S, Boland CR, Llor X (2010) Aberrant DNA methylation in hereditary nonpolyposis colorectal cancer without mismatch repair deficiency. Gastroenterology 138(5):1854–1862. doi:10.1053/j.gastro.2010.01.035
Gonzalo S (2010) Epigenetic alterations in aging. J Appl Physiol 109(2):586–597. doi:10.1152/japplphysiol.00238.2010
Graham T, Boissinot S (2006) The genomic distribution of L1 elements: the role of insertion bias and natural selection. J Biomed Biotechnol 2006(1):75327. doi:10.1155/JBB/2006/75327
Han JS, Szak ST, Boeke JD (2004) Transcriptional disruption by the L1 retrotransposon and implications for mammalian transcriptomes. Nature 429(6989):268–274. doi:10.1038/nature02536
Hansen RS, Wijmenga C, Luo P, Stanek AM, Canfield TK, Weemaes CM, Gartler SM (1999) The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome. Proc Natl Acad Sci USA 96(25):14412–14417
Hazra A, Fuchs CS, Kawasaki T, Kirkner GJ, Hunter DJ, Ogino S (2010) Germline polymorphisms in the one-carbon metabolism pathway and DNA methylation in colorectal cancer. Cancer Causes Control 21(3):331–345. doi:10.1007/s10552-009-9464-2
Hou L, Wang H, Sartori S, Gawron A, Lissowska J, Bollati V, Tarantini L, Zhang FF, Zatonski W, Chow WH, Baccarelli A (2010) Blood leukocyte DNA hypomethylation and gastric cancer risk in a high-risk Polish population. Int J Cancer 127(8):1866–1874. doi:10.1002/ijc.25190
Hsiung DT, Marsit CJ, Houseman EA, Eddy K, Furniss CS, McClean MD, Kelsey KT (2007) Global DNA methylation level in whole blood as a biomarker in head and neck squamous cell carcinoma. Cancer Epidemiol Biomark Prev 16(1):108–114. doi:10.1158/1055-9965.EPI-06-0636
Huang CR, Schneider AM, Lu Y, Niranjan T, Shen P, Robinson MA, Steranka JP, Valle D, Civin CI, Wang T, Wheelan SJ, Ji H, Boeke JD, Burns KH (2010) Mobile interspersed repeats are major structural variants in the human genome. Cell 141(7):1171–1182. doi:10.1016/j.cell.2010.05.026
Iacopetta B, Grieu F, Phillips M, Ruszkiewicz A, Moore J, Minamoto T, Kawakami K (2007) Methylation levels of LINE-1 repeats and CpG island loci are inversely related in normal colonic mucosa. Cancer Sci 98(9):1454–1460. doi:10.1111/j.1349-7006.2007.00548.x
Ibrahim AE, Arends MJ, Silva AL, Wyllie AH, Greger L, Ito Y, Vowler SL, Huang TH, Tavare S, Murrell A, Brenton JD (2011) Sequential DNA methylation changes are associated with DNMT3B overexpression in colorectal neoplastic progression. Gut 60(4):499–508. doi:10.1136/gut.2010.223602
Igarashi S, Suzuki H, Niinuma T, Shimizu H, Nojima M, Iwaki H, Nobuoka T, Nishida T, Miyazaki Y, Takamaru H, Yamamoto E, Yamamoto H, Tokino T, Hasegawa T, Hirata K, Imai K, Toyota M, Shinomura Y (2010) A novel correlation between LINE-1 hypomethylation and the malignancy of gastrointestinal stromal tumors. Clin Cancer Res 16(21):5114–5123. doi:10.1158/1078-0432.CCR-10-0581
Irahara N, Nosho K, Baba Y, Shima K, Lindeman NI, Hazra A, Schernhammer ES, Hunter DJ, Fuchs CS, Ogino S (2010) Precision of pyrosequencing assay to measure LINE-1 methylation in colon cancer, normal colonic mucosa, and peripheral blood cells. J Mol Diagn 12(2):177–183. doi:10.2353/jmoldx.2010.090106
Iramaneerat K, Rattanatunyong P, Khemapech N, Triratanachat S, Mutirangura A (2011) HERV-K hypomethylation in ovarian clear cell carcinoma is associated with a poor prognosis and platinum resistance. Int J Gynecol Cancer 21(1):51–57. doi:10.1097/IGC.0b013e3182021c1a
Iskow RC, McCabe MT, Mills RE, Torene S, Pittard WS, Neuwald AF, Van Meir EG, Vertino PM, Devine SE (2010) Natural mutagenesis of human genomes by endogenous retrotransposons. Cell 141(7):1253–1261. doi:10.1016/j.cell.2010.05.020
Ji W, Hernandez R, Zhang XY, Qu GZ, Frady A, Varela M, Ehrlich M (1997) DNA demethylation and pericentromeric rearrangements of chromosome 1. Mutat Res 379(1):33–41. doi:S0027-5107(97)00088-2
Jin M, Kawakami K, Fukui Y, Tsukioka S, Oda M, Watanabe G, Takechi T, Oka T, Minamoto T (2009) Different histological types of non-small cell lung cancer have distinct folate and DNA methylation levels. Cancer Sci 100(12):2325–2330. doi:10.1111/j.1349-7006.2009.01321.x
Jintaridth P, Mutirangura A (2011) Distinctive patterns of age-dependent hypomethylation in interspersed repetitive sequences. Physiol Genomics 41(2):194–200. doi:10.1152/physiolgenomics.00146.2009
Juhlin CC, Kiss NB, Villablanca A, Haglund F, Nordenstrom J, Hoog A, Larsson C (2010) Frequent promoter hypermethylation of the APC and RASSF1A tumour suppressors in parathyroid tumours. PLoS ONE 5(3):e9472. doi:10.1371/journal.pone.0009472
Jurgens B, Schmitz-Drager BJ, Schulz WA (1996) Hypomethylation of L1 LINE sequences prevailing in human urothelial carcinoma. Cancer Res 56(24):5698–5703
Kawakami K, Matsunoki A, Kaneko M, Saito K, Watanabe G, Minamoto T (2011) Long interspersed nuclear element-1 hypomethylation is a potential biomarker for the prediction of response to oral fluoropyrimidines in microsatellite stable and CpG island methylator phenotype-negative colorectal cancer. Cancer Sci 102(1):166–174. doi:10.1111/j.1349-7006.2010.01776.x
Kim WJ, Vo QN, Shrivastav M, Lataxes TA, Brown KD (2002) Aberrant methylation of the ATM promoter correlates with increased radiosensitivity in a human colorectal tumor cell line. Oncogene 21(24):3864–3871. doi:10.1038/sj.onc.1205485
Kim BH, Cho NY, Shin SH, Kwon HJ, Jang JJ, Kang GH (2009a) CpG island hypermethylation and repetitive DNA hypomethylation in premalignant lesion of extrahepatic cholangiocarcinoma. Virchows Arch 455(4):343–351. doi:10.1007/s00428-009-0829-4
Kim MJ, White-Cross JA, Shen L, Issa JP, Rashid A (2009b) Hypomethylation of long interspersed nuclear element-1 in hepatocellular carcinomas. Mod Pathol 22(3):442–449. doi:10.1038/modpathol.2008.203
Kim SJ, Kelly WK, Fu A, Haines K, Hoffman A, Zheng T, Zhu Y (2011) Genome-wide methylation analysis identifies involvement of TNF-alpha mediated cancer pathways in prostate cancer. Cancer Lett 302(1):47–53. doi:10.1016/j.canlet.2010.12.010
Kindich R, Florl AR, Kamradt J, Lehmann J, Muller M, Wullich B, Schulz WA (2006) Relationship of NKX3.1 and MYC gene copy number ratio and DNA hypomethylation to prostate carcinoma stage. Eur Urol 49(1):169–175. doi:10.1016/j.eururo.2005.09.012, discussion 175
Kitkumthorn N, Mutirangura A (2010) LINE-1 methylation difference between ameloblastoma and keratocystic odontogenic tumor. Oral Dis 16(3):286–291. doi:10.1111/j.1601-0825.2009.01640.x
Kongruttanachok N, Phuangphairoj C, Thongnak A, Ponyeam W, Rattanatanyong P, Pornthanakasem W, Mutirangura A (2010) Replication independent DNA double-strand break retention may prevent genomic instability. Mol Cancer 9:70. doi:10.1186/1476-4598-9-70
Kremenskoy M, Kremenska Y, Ohgane J, Hattori N, Tanaka S, Hashizume K, Shiota K (2003) Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses. Biochem Biophys Res Commun 311(4):884–890. doi:S0006291X03021739
Kwon HJ, Kim JH, Bae JM, Cho NY, Kim TY, Kang GH (2010) DNA methylation changes in ex-adenoma carcinoma of the large intestine. Virchows Arch 457(4):433–441. doi:10.1007/s00428-010-0958-9
Laird PW (2010) Principles and challenges of genome-wide DNA methylation analysis. Nat Rev Genet 11(3):191–203. doi:10.1038/nrg2732
Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, Howland J, Kann L, Lehoczky J, LeVine R, McEwan P, McKernan K, Meldrim J, Mesirov JP, Miranda C, Morris W, Naylor J, Raymond C, Rosetti M, Santos R, Sheridan A, Sougnez C, Stange-Thomann N, Stojanovic N, Subramanian A, Wyman D, Rogers J, Sulston J, Ainscough R, Beck S, Bentley D, Burton J, Clee C, Carter N, Coulson A, Deadman R, Deloukas P, Dunham A, Dunham I, Durbin R, French L, Grafham D, Gregory S, Hubbard T, Humphray S, Hunt A, Jones M, Lloyd C, McMurray A, Matthews L, Mercer S, Milne S, Mullikin JC, Mungall A, Plumb R, Ross M, Shownkeen R, Sims S, Waterston RH, Wilson RK, Hillier LW, McPherson JD, Marra MA, Mardis ER, Fulton LA, Chinwalla AT, Pepin KH, Gish WR, Chissoe SL, Wendl MC, Delehaunty KD, Miner TL, Delehaunty A, Kramer JB, Cook LL, Fulton RS, Johnson DL, Minx PJ, Clifton SW, Hawkins T, Branscomb E, Predki P, Richardson P, Wenning S, Slezak T, Doggett N, Cheng JF, Olsen A, Lucas S, Elkin C, Uberbacher E, Frazier M, Gibbs RA, Muzny DM, Scherer SE, Bouck JB, Sodergren EJ, Worley KC, Rives CM, Gorrell JH, Metzker ML, Naylor SL, Kucherlapati RS, Nelson DL, Weinstock GM, Sakaki Y, Fujiyama A, Hattori M, Yada T, Toyoda A, Itoh T, Kawagoe C, Watanabe H, Totoki Y, Taylor T, Weissenbach J, Heilig R, Saurin W, Artiguenave F, Brottier P, Bruls T, Pelletier E, Robert C, Wincker P, Smith DR, Doucette-Stamm L, Rubenfield M, Weinstock K, Lee HM, Dubois J, Rosenthal A, Platzer M, Nyakatura G, Taudien S, Rump A, Yang H, Yu J, Wang J, Huang G, Gu J, Hood L, Rowen L, Madan A, Qin S, Davis RW, Federspiel NA, Abola AP, Proctor MJ, Myers RM, Schmutz J, Dickson M, Grimwood J, Cox DR, Olson MV, Kaul R, Shimizu N, Kawasaki K, Minoshima S, Evans GA, Athanasiou M, Schultz R, Roe BA, Chen F, Pan H, Ramser J, Lehrach H, Reinhardt R, McCombie WR, de la Bastide M, Dedhia N, Blocker H, Hornischer K, Nordsiek G, Agarwala R, Aravind L, Bailey JA, Bateman A, Batzoglou S, Birney E, Bork P, Brown DG, Burge CB, Cerutti L, Chen HC, Church D, Clamp M, Copley RR, Doerks T, Eddy SR, Eichler EE, Furey TS, Galagan J, Gilbert JG, Harmon C, Hayashizaki Y, Haussler D, Hermjakob H, Hokamp K, Jang W, Johnson LS, Jones TA, Kasif S, Kaspryzk A, Kennedy S, Kent WJ, Kitts P, Koonin EV, Korf I, Kulp D, Lancet D, Lowe TM, McLysaght A, Mikkelsen T, Moran JV, Mulder N, Pollara VJ, Ponting CP, Schuler G, Schultz J, Slater G, Smit AF, Stupka E, Szustakowski J, Thierry-Mieg D, Thierry-Mieg J, Wagner L, Wallis J, Wheeler R, Williams A, Wolf YI, Wolfe KH, Yang SP, Yeh RF, Collins F, Guyer MS, Peterson J, Felsenfeld A, Wetterstrand KA, Patrinos A, Morgan MJ, de Jong P, Catanese JJ, Osoegawa K, Shizuya H, Choi S, Chen YJ (2001) Initial sequencing and analysis of the human genome. Nature 409(6822):860–921
Lee JJ, Geli J, Larsson C, Wallin G, Karimi M, Zedenius J, Hoog A, Foukakis T (2008) Gene-specific promoter hypermethylation without global hypomethylation in follicular thyroid cancer. Int J Oncol 33(4):861–869
Lee HS, Kim BH, Cho NY, Yoo EJ, Choi M, Shin SH, Jang JJ, Suh KS, Kim YS, Kang GH (2009) Prognostic implications of and relationship between CpG island hypermethylation and repetitive DNA hypomethylation in hepatocellular carcinoma. Clin Cancer Res 15(3):812–820. doi:10.1158/1078-0432.CCR-08-0266
Lu LJ, Randerath K (1984) Long term instability and molecular mechanism of 5-azacytidine-induced DNA hypomethylation in normal and neoplastic tissues in vivo. Mol Pharmacol 26(3):594–603
Lupski JR (2010) Retrotransposition and structural variation in the human genome. Cell 141(7):1110–1112. doi:10.1016/j.cell.2010.06.014
Lutz D, Lowel M, Kroger H, Kubsch D, Uecker W (1972) In vivo methylation of DNA in different organs of rat at various ages. Z Naturforsch B 27(8):992–995
Maraschio P, Zuffardi O, Dalla Fior T, Tiepolo L (1988) Immunodeficiency, centromeric heterochromatin instability of chromosomes 1, 9, and 16, and facial anomalies: the ICF syndrome. J Med Genet 25(3):173–180
Matlik K, Redik K, Speek M (2006) L1 antisense promoter drives tissue-specific transcription of human genes. J Biomed Biotechnol 2006(1):71753. doi:10.1155/JBB/2006/71753
Matsubayashi H, Skinner HG, Iacobuzio-Donahue C, Abe T, Sato N, Riall TS, Yeo CJ, Kern SE, Goggins M (2005) Pancreaticobiliary cancers with deficient methylenetetrahydrofolate reductase genotypes. Clin Gastroenterol Hepatol 3(8):752–760. doi:S1542-3565(05)00359-9
Matsuzaki K, Deng G, Tanaka H, Kakar S, Miura S, Kim YS (2005) The relationship between global methylation level, loss of heterozygosity, and microsatellite instability in sporadic colorectal cancer. Clin Cancer Res 11(24 Pt 1):8564–8569. doi:10.1158/1078-0432.CCR-05-0859
Mavrou A, Tsangaris GT, Roma E, Kolialexi A (2008) The ATM gene and ataxia telangiectasia. Anticancer Res 28(1B):401–405
Menendez L, Benigno BB, McDonald JF (2004) L1 and HERV-W retrotransposons are hypomethylated in human ovarian carcinomas. Mol Cancer 3:12. doi:10.1186/1476-4598-3-12
Migeon BR, Holland MM, Driscoll DJ, Robinson JC (1991) Programmed demethylation in CpG islands during human fetal development. Somat Cell Mol Genet 17(2):159–168
Miki Y, Nishisho I, Horii A, Miyoshi Y, Utsunomiya J, Kinzler KW, Vogelstein B, Nakamura Y (1992) Disruption of the APC gene by a retrotransposal insertion of L1 sequence in a colon cancer. Cancer Res 52(3):643–645
Moran JV (1999) Human L1 retrotransposition: insights and peculiarities learned from a cultured cell retrotransposition assay. Genetica 107(1–3):39–51
Moran JV, DeBerardinis RJ, Kazazian HH Jr (1999) Exon shuffling by L1 retrotransposition. Science 283(5407):1530–1534
Neuhausen A, Florl AR, Grimm MO, Schulz WA (2006) DNA methylation alterations in urothelial carcinoma. Cancer Biol Ther 5(8):993–1001. doi:2885
Nosho K, Kure S, Irahara N, Shima K, Baba Y, Spiegelman D, Meyerhardt JA, Giovannucci EL, Fuchs CS, Ogino S (2009a) A prospective cohort study shows unique epigenetic, genetic, and prognostic features of synchronous colorectal cancers. Gastroenterology 137(5):1609–1620. doi:10.1053/j.gastro.2009.08.002, e1601–1603
Nosho K, Shima K, Irahara N, Kure S, Baba Y, Kirkner GJ, Chen L, Gokhale S, Hazra A, Spiegelman D, Giovannucci EL, Jaenisch R, Fuchs CS, Ogino S (2009b) DNMT3B expression might contribute to CpG island methylator phenotype in colorectal cancer. Clin Cancer Res 15(11):3663–3671. doi:10.1158/1078-0432.CCR-08-2383
Ogino S, Kawasaki T, Nosho K, Ohnishi M, Suemoto Y, Kirkner GJ, Fuchs CS (2008a) LINE-1 hypomethylation is inversely associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer. Int J Cancer 122(12):2767–2773. doi:10.1002/ijc.23470
Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernhammer ES, Giovannucci EL, Fuchs CS (2008b) A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst 100(23):1734–1738. doi:10.1093/jnci/djn359
Park SY, Yoo EJ, Cho NY, Kim N, Kang GH (2009) Comparison of CpG island hypermethylation and repetitive DNA hypomethylation in premalignant stages of gastric cancer, stratified for Helicobacter pylori infection. J Pathol 219(4):410–416. doi:10.1002/path.2596
Pattamadilok J, Huapai N, Rattanatanyong P, Vasurattana A, Triratanachat S, Tresukosol D, Mutirangura A (2008) LINE-1 hypomethylation level as a potential prognostic factor for epithelial ovarian cancer. Int J Gynecol Cancer 18(4):711–717. doi:10.1111/j.1525-1438.2007.01117.x
Penzkofer T, Dandekar T, Zemojtel T (2005) L1Base: from functional annotation to prediction of active LINE-1 elements. Nucleic Acids Res 33(Database issue):D498–500
Perrin D, Ballestar E, Fraga MF, Frappart L, Esteller M, Guerin JF, Dante R (2007) Specific hypermethylation of LINE-1 elements during abnormal overgrowth and differentiation of human placenta. Oncogene 26(17):2518–2524. doi:10.1038/sj.onc.1210039
Phokaew C, Kowudtitham S, Subbalekha K, Shuangshoti S, Mutirangura A (2008) LINE-1 methylation patterns of different loci in normal and cancerous cells. Nucleic Acids Res 36(17):5704–5712. doi:10.1093/nar/gkn571
Pobsook T, Subbalekha K, Sannikorn P, Mutirangura A (2011) Improved measurement of LINE-1 sequence methylation for cancer detection. Clin Chim Acta 412(3–4):314–321. doi:10.1016/j.cca.2010.10.030
Pornthanakasem W, Kongruttanachok N, Phuangphairoj C, Suyarnsestakorn C, Sanghangthum T, Oonsiri S, Ponyeam W, Thanasupawat T, Matangkasombut O, Mutirangura A (2008) LINE-1 methylation status of endogenous DNA double-strand breaks. Nucleic Acids Res 36(11):3667–3675. doi:10.1093/nar/gkn261
Rangwala SH, Zhang L, Kazazian HH Jr (2009) Many LINE1 elements contribute to the transcriptome of human somatic cells. Genome Biol 10(9):R100. doi:10.1186/gb-2009-10-9-r100
Richardson B, Scheinbart L, Strahler J, Gross L, Hanash S, Johnson M (1990) Evidence for impaired T cell DNA methylation in systemic lupus erythematosus and rheumatoid arthritis. Arthritis Rheum 33(11):1665–1673
Rodriguez J, Vives L, Jorda M, Morales C, Munoz M, Vendrell E, Peinado MA (2008) Genome-wide tracking of unmethylated DNA Alu repeats in normal and cancer cells. Nucleic Acids Res 36(3):770–784. doi:10.1093/nar/gkm1105
Roman-Gomez J, Jimenez-Velasco A, Agirre X, Cervantes F, Sanchez J, Garate L, Barrios M, Castillejo JA, Navarro G, Colomer D, Prosper F, Heiniger A, Torres A (2005) Promoter hypomethylation of the LINE-1 retrotransposable elements activates sense/antisense transcription and marks the progression of chronic myeloid leukemia. Oncogene 24(48):7213–7223. doi:10.1038/sj.onc.1208866
Roman-Gomez J, Jimenez-Velasco A, Agirre X, Castillejo JA, Navarro G, San Jose-Eneriz E, Garate L, Cordeu L, Cervantes F, Prosper F, Heiniger A, Torres A (2008) Repetitive DNA hypomethylation in the advanced phase of chronic myeloid leukemia. Leuk Res 32(3):487–490. doi:10.1016/j.leukres.2007.07.021
Romermann D, Hasemeier B, Metzig K, Schlegelberger B, Langer F, Kreipe H, Lehmann U (2007) Methylation status of LINE-1 sequences in patients with MDS or secondary AML. Verh Dtsch Ges Pathol 91:338–342
Saito K, Kawakami K, Matsumoto I, Oda M, Watanabe G, Minamoto T (2010) Long interspersed nuclear element 1 hypomethylation is a marker of poor prognosis in stage IA non-small cell lung cancer. Clin Cancer Res 16(8):2418–2426. doi:10.1158/1078-0432.CCR-09-2819
Santourlidis S, Florl A, Ackermann R, Wirtz HC, Schulz WA (1999) High frequency of alterations in DNA methylation in adenocarcinoma of the prostate. Prostate 39(3):166–174. doi:10.1002/(SICI)1097-0045(19990515)39:3<166::AID-PROS4>3.0.CO;2-J
Sassaman DM, Dombroski BA, Moran JV, Kimberland ML, Naas TP, DeBerardinis RJ, Gabriel A, Swergold GD, Kazazian HH Jr (1997) Many human L1 elements are capable of retrotransposition. Nat Genet 16(1):37–43. doi:10.1038/ng0597-37
Schulz WA, Elo JP, Florl AR, Pennanen S, Santourlidis S, Engers R, Buchardt M, Seifert HH, Visakorpi T (2002) Genomewide DNA hypomethylation is associated with alterations on chromosome 8 in prostate carcinoma. Genes Chromosom Cancer 35(1):58–65. doi:10.1002/gcc.10092
Schulz WA, Steinhoff C, Florl AR (2006) Methylation of endogenous human retroelements in health and disease. Curr Top Microbiol Immunol 310:211–250
Shuangshoti S, Hourpai N, Pumsuk U, Mutirangura A (2007) Line-1 hypomethylation in multistage carcinogenesis of the uterine cervix. Asian Pac J Cancer Prev 8(2):307–309
Smith IM, Mydlarz WK, Mithani SK, Califano JA (2007) DNA global hypomethylation in squamous cell head and neck cancer associated with smoking, alcohol consumption and stage. Int J Cancer 121(8):1724–1728. doi:10.1002/ijc.22889
Sonpavde G, Aparicio AM, Zhan F, North B, Delaune R, Garbo LE, Rousey SR, Weinstein RE, Xiao L, Boehm KA, Asmar L, Fleming MT, Galsky MD, Berry WR, Von Hoff DD (2009) Azacitidine favorably modulates PSA kinetics correlating with plasma DNA LINE-1 hypomethylation in men with chemonaive castration-resistant prostate cancer. Urol Oncol. doi:10.1016/j.urolonc.2009.09.015
Speek M (2001) Antisense promoter of human L1 retrotransposon drives transcription of adjacent cellular genes. Mol Cell Biol 21(6):1973–1985. doi:10.1128/MCB.21.6.1973-1985.2001
Subbalekha K, Pimkhaokham A, Pavasant P, Chindavijak S, Phokaew C, Shuangshoti S, Matangkasombut O, Mutirangura A (2009) Detection of LINE-1s hypomethylation in oral rinses of oral squamous cell carcinoma patients. Oral Oncol 45(2):184–191. doi:10.1016/j.oraloncology.2008.05.002
Suter CM, Martin DI, Ward RL (2004) Hypomethylation of L1 retrotransposons in colorectal cancer and adjacent normal tissue. Int J Colorectal Dis 19(2):95–101. doi:10.1007/s00384-003-0539-3
Suyarnsestakorn C, Thanasupawat T, Leelahavanichkul K, Gutkind JS, Mutirangura A (2010) Ataxia telangiectasia mutated nuclear localization in head and neck cancer cells is PPP2R2B-dependent. Asian Biomed 4(3):373–383
Szpakowski S, Sun X, Lage JM, Dyer A, Rubinstein J, Kowalski D, Sasaki C, Costa J, Lizardi PM (2009) Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements. Gene 448(2):151–167. doi:10.1016/j.gene.2009.08.006
Takai D, Yagi Y, Habib N, Sugimura T, Ushijima T (2000) Hypomethylation of LINE1 retrotransposon in human hepatocellular carcinomas, but not in surrounding liver cirrhosis. Jpn J Clin Oncol 30(7):306–309
Tangkijvanich P, Hourpai N, Rattanatanyong P, Wisedopas N, Mahachai V, Mutirangura A (2007) Serum LINE-1 hypomethylation as a potential prognostic marker for hepatocellular carcinoma. Clin Chim Acta 379(1–2):127–133. doi:10.1016/j.cca.2006.12.029
Tellez CS, Shen L, Estecio MR, Jelinek J, Gershenwald JE, Issa JP (2009) CpG island methylation profiling in human melanoma cell lines. Melanoma Res 19(3):146–155
Trankenschuh W, Puls F, Christgen M, Albat C, Heim A, Poczkaj J, Fleming P, Kreipe H, Lehmann U (2010) Frequent and distinct aberrations of DNA methylation patterns in fibrolamellar carcinoma of the liver. PLoS ONE 5(10):e13688. doi:10.1371/journal.pone.0013688
Vaissiere T, Cuenin C, Paliwal A, Vineis P, Hoek G, Krzyzanowski M, Airoldi L, Dunning A, Garte S, Hainaut P, Malaveille C, Overvad K, Clavel-Chapelon F, Linseisen J, Boeing H, Trichopoulou A, Trichopoulos D, Kaladidi A, Palli D, Krogh V, Tumino R, Panico S, Bueno-De-Mesquita HB, Peeters PH, Kumle M, Gonzalez CA, Martinez C, Dorronsoro M, Barricarte A, Navarro C, Quiros JR, Berglund G, Janzon L, Jarvholm B, Day NE, Key TJ, Saracci R, Kaaks R, Riboli E, Herceg Z (2009) Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids. Epigenetics 4(4):221–230. doi:8833
Wang L, Wang F, Guan J, Le J, Wu L, Zou J, Zhao H, Pei L, Zheng X, Zhang T (2010) Relation between hypomethylation of long interspersed nucleotide elements and risk of neural tube defects. Am J Clin Nutr 91(5):1359–1367. doi:10.3945/ajcn.2009.28858
Watanabe Y, Maekawa M (2010) Methylation of DNA in cancer. Adv Clin Chem 52:145–167
Watts GS, Futscher BW, Holtan N, Degeest K, Domann FE, Rose SL (2008) DNA methylation changes in ovarian cancer are cumulative with disease progression and identify tumor stage. BMC Med Genomics 1:47. doi:10.1186/1755-8794-1-47
Weber B, Kimhi S, Howard G, Eden A, Lyko F (2010) Demethylation of a LINE-1 antisense promoter in the cMet locus impairs Met signalling through induction of illegitimate transcription. Oncogene 29(43):5775–5784. doi:10.1038/onc.2010.227
Weisenberger DJ, Campan M, Long TI, Kim M, Woods C, Fiala E, Ehrlich M, Laird PW (2005) Analysis of repetitive element DNA methylation by MethyLight. Nucleic Acids Res 33(21):6823–6836. doi:10.1093/nar/gki987
Wilhelm CS, Kelsey KT, Butler R, Plaza S, Gagne L, Zens MS, Andrew AS, Morris S, Nelson HH, Schned AR, Karagas MR, Marsit CJ (2010) Implications of LINE1 methylation for bladder cancer risk in women. Clin Cancer Res 16(5):1682–1689. doi:10.1158/1078-0432.CCR-09-2983
Wolff EM, Byun HM, Han HF, Sharma S, Nichols PW, Siegmund KD, Yang AS, Jones PA, Liang G (2010) Hypomethylation of a LINE-1 promoter activates an alternate transcript of the MET oncogene in bladders with cancer. PLoS Genet 6(4):e1000917. doi:10.1371/journal.pgen.1000917
Woloszynska-Read A, Mhawech-Fauceglia P, Yu J, Odunsi K, Karpf AR (2008) Intertumor and intratumor NY-ESO-1 expression heterogeneity is associated with promoter-specific and global DNA methylation status in ovarian cancer. Clin Cancer Res 14(11):3283–3290. doi:10.1158/1078-0432.CCR-07-5279
Woloszynska-Read A, Zhang W, Yu J, Link PA, Mhawech-Fauceglia P, Collamat G, Akers SN, Ostler KR, Godley LA, Odunsi KO, Karpf AR (2011) Coordinated cancer germline antigen promoter and global DNA hypomethylation in ovarian cancer: association with BORIS/CTCF expression ratio and advanced stage. Clin Cancer Res. doi:10.1158/1078-0432.CCR-10-2315
Xiang S, Liu Z, Zhang B, Zhou J, Zhu BD, Ji J, Deng D (2010) Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas. BMC Cancer 10:44. doi:10.1186/1471-2407-10-44
Xie H, Wang M, Bonaldo Mde F, Smith C, Rajaram V, Goldman S, Tomita T, Soares MB (2009) High-throughput sequence-based epigenomic analysis of Alu repeats in human cerebellum. Nucleic Acids Res 37(13):4331–4340. doi:10.1093/nar/gkp393
Xie H, Wang M, Bonaldo Mde F, Rajaram V, Stellpflug W, Smith C, Arndt K, Goldman S, Tomita T, Soares MB (2010) Epigenomic analysis of Alu repeats in human ependymomas. Proc Natl Acad Sci USA 107(15):6952–6957. doi:10.1073/pnas.0913836107
Xie H, Wang M, de Andrade A, Bonaldo MD, Galat V, Arndt K, Rajaram V, Goldman S, Tomita T, Soares MB (2011) Genome-wide quantitative assessment of variation in DNA methylation patterns. Nucleic Acids Res 1(1):1–10. doi:10.1093/nar/gkr017
Xiong Z, Laird PW (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25(12):2532–2534. doi:gka376
Yang AS, Estecio MR, Doshi K, Kondo Y, Tajara EH, Issa JP (2004) A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Res 32(3):e38. doi:10.1093/nar/gnh032
Yegnasubramanian S, Haffner MC, Zhang Y, Gurel B, Cornish TC, Wu Z, Irizarry RA, Morgan J, Hicks J, DeWeese TL, Isaacs WB, Bova GS, De Marzo AM, Nelson WG (2008) DNA hypomethylation arises later in prostate cancer progression than CpG island hypermethylation and contributes to metastatic tumor heterogeneity. Cancer Res 68(21):8954–8967. doi:10.1158/0008-5472.CAN-07-6088
Yoo EJ, Park SY, Cho NY, Kim N, Lee HS, Kang GH (2008) Helicobacter pylori-infection-associated CpG island hypermethylation in the stomach and its possible association with polycomb repressive marks. Virchows Arch 452(5):515–524. doi:10.1007/s00428-008-0596-7
Yoshida T, Yamashita S, Takamura-Enya T, Niwa T, Ando T, Enomoto S, Maekita T, Nakazawa K, Tatematsu M, Ichinose M, Ushijima T (2011) Alu and Satalpha hypomethylation in Helicobacter pylori-infected gastric mucosae. Int J Cancer 128(1):33–39. doi:10.1002/ijc.25534
References in Thailand have been supported by the Thailand Research Fund, the National Center for Biotechnology and Genetic Engineering (Thailand), Four Seasons Hotel Bangkok and the Thai Red Cross Society 4th Cancer Care Charity, a RF-MRG Scientific Researcher grant, and Chulalongkorn University.
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The authors have no conflict of interest to report.
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Kitkumthorn, N., Mutirangura, A. Long interspersed nuclear element-1 hypomethylation in cancer: biology and clinical applications. Clin Epigenet 2, 315–330 (2011) doi:10.1007/s13148-011-0032-8
- Long interspersed nuclear element-1s
- DNA methylation
- Partial methylation