Apiwat Mutirangura

Distinctive pattern of LINE-1 methylation level in normal tissues and the association with carcinogenesis

Genome-wide losses of DNA methylation have been regarded as a common epigenetic event in malignancies and may play crucial roles in carcinogenesis. Limited information is available on the global methylation status in normal tissues and other cancer types beyond colonic carcinoma. Here we applied the combined bisulfite restriction analysis PCR to evaluate the methylation status of LINE-1 repetitive sequences in genomic DNA derived from microdissected samples from several human normal and neoplastic tissues. We found that methylation of LINE-1 in leukocytes was independent of age and gender. In contrast, normal tissues from different organs showed tissue-specific levels of methylated LINE-1. Globally, most carcinomas including breast, colon, lung, head and neck, bladder, esophagus, liver, prostate, and stomach, revealed a greater percentage of hypomethylation than their normal tissue counterparts. Furthermore, DNA derived from sera of patients with carcinoma displayed more LINE-1 hypomethylation than those of noncarcinoma individuals. Finally, in a colonic carcinogenesis model, we detected significantly greater hypomethylation in carcinoma than those of dysplastic polyp and histological normal colonic epithelium. Thus, the methylation status is a unique feature of a specific tissue type and the global hypomethylation is a common epigenetic process in cancer, which may progressively evolve during multistage carcinogenesis.

Molecular dissection of the Prader-Willi/Angelman syndrome region (15q11–13) by YAC cloning and FISH analysis

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct mental retardation disorders associated with deletions of proximal 15q (q11-q13) of different parental origin. Yeast artificial chromosome (YAC) clones were isolated for 9 previously mapped DNA probes from this region, and for one newly derived marker, LS6-1 (D15S113). A YAC contig of 1-1.5 Mb encompassing four markers (ML34, IR4-3R, PW71, and TD189-1) was constructed. Multi-color fluorescence in situ hybridization (FISH) analysis of interphase nuclei was combined with YAC contig information to provide the following order of markers: cen-IR39-ML34-IR4-3R-PW71-TD189-1-LS6++ +-1-TD3-21-GABRB3-IR10-1-CMW1-tel. FISH analysis was performed on 8 cases of PWS and 3 cases of AS, including 5 patients with normal karyotypes. All eleven patients were deleted for YACs in the interval from IR4-3R to GABRB3. On the proximal side of the deletion interval, 10/10 breakpoints fell within a single ML34 YAC of 370 kb. On the distal side, 8/9 breakpoints fell within a single IR10-1 YAC of 200 kb. These results indicate a striking consistency in the location of the proximal and distal breakpoints in PWS and AS patients. FISH analysis on a previously reported case of familial AS confirmed a submicroscopic deletion including YACs corresponding to LS6-1, TD3-21 and GABRB3 and supports the separation of the PWS and AS critical regions. Since these three YACs do not overlap each other, the minimum size of the AS critical region is > or = 650 kb.

Distinctive patterns of age-dependent hypomethylation in interspersed repetitive sequences

Interspersed repetitive sequences (IRSs) are a major contributor to genome size and may contribute to cellular functions. IRSs are subdivided according to size and functionally related structures into short interspersed elements, long interspersed elements (LINEs), DNA transposons, and LTR-retrotransposons. Many IRSs may produce RNA and regulate genes by a variety of mechanisms. The majority of DNA methylation occurs in IRSs and is believed to suppress IRS activities. Global hypomethylation, or the loss of genome-wide methylation, is a common epigenetic event not only in senescent cells but also in cancer cells. Loss of LINE-1 methylation has been characterized in many cancers. Here, we evaluated the methylation levels of peripheral blood mononuclear cells of LINE-1, Alu, and human endogenous retrovirus K (HERV-K) in 177 samples obtained from volunteers between 20 and 88 yr of age. Age was negatively associated with methylation levels of Alu (r = -0.452, P < 10(-3)) and HERV-K (r = -0.326, P < 10(-3)) but not LINE-1 (r = 0.145, P = 0.055). Loss of methylation of Alu occurred during ages 34-68 yr, and loss of methylation of HERV-K occurred during ages 40-63 yr and again during ages 64-83 yr. Interestingly, methylation of Alu and LINE-1 are directly associated, particularly at ages 49 yr and older (r = 0.49, P < 10(-3)). Therefore, only some types of IRSs lose methylation at certain ages. Moreover, Alu and HERV-K become hypomethylated differently. Finally, there may be several mechanisms of global methylation. However, not all of these mechanisms are age-dependent. This finding may lead to a better understanding of not only the biological causes and consequences of genome-wide hypomethylation but also the role of IRSs in the aging process.

Long interspersed nuclear element-1 hypomethylation in cancer: biology and clinical applications

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.

Human papillomavirus DNA in plasma of patients with cervical cancer

BackgroundHuman papillomavirus (HPV) is a crucial etiological factor for cervical cancer (CC) development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients.ResultsWhereas 6 out of 50 (12%) HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67). Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA.ConclusionsThe plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA.

A complete YAC contig of the Prader-Willi/Angelman chromosome region (15q11-q13) and refined localization of the SNRPN gene.

Since a previous report of a partial YAC contig of the Prader-Willi/Angelman chromosome region (15q11-q13), a complete contig spanning approximately 3.5 Mb has been developed. YACs were isolated from two human genomic libraries by PCR and hybridization screening methods. Twenty-three sequence-tagged sites (STSs) were mapped within the contig, a density of approximately 1 per 200 kb. Overlaps between YAC clones were identified by Alu-PCR dot-blot analysis and confirmed by STS mapping or hybridization with ends of YAC inserts. The gene encoding small nuclear ribonucleoprotein-associated peptide N (SNRPN), recently identified as a candidate gene for Prader-Willi syndrome, was localized within this contig between markers PW71 and TD3-21. Loci mapped within and immediately flanking the Prader-Willi/Angelman chromosome region contig are ordered as follows: cen-IR39-ML34-IR4-3R-TD189-1-PW71-SNRPN -TD3-21- LS6-1-GABRB3,D15S97-GABRA5-IR10-1-CMW1+ ++-tel. This YAC contig will be a useful resource for more detailed physical mapping of the region, for generation of new DNA markers, and for mapping or cloning candidate genes for the Prader-Willi and Angelman syndromes.

LINE-1 methylation patterns of different loci in normal and cancerous cells

This study evaluated methylation patterns of long interspersed nuclear element-1 (LINE-1) sequences from 17 loci in several cell types, including squamous cell cancer cell lines, normal oral epithelium (NOE), white blood cells and head and neck squamous cell cancers (HNSCC). Although sequences of each LINE-1 are homologous, LINE-1 methylation levels at each locus are different. Moreover, some loci demonstrate the different methylation levels between normal tissue types. Interestingly, in some chromosomal regions, wider ranges of LINE-1 methylation levels were observed. In cancerous cells, the methylation levels of most LINE-1 loci demonstrated a positive correlation with each other and with the genome-wide levels. Therefore, the loss of genome-wide methylation in cancerous cells occurs as a generalized process. However, different LINE-1 loci showed different incidences of HNSCC hypomethylation, which is a lower methylation level than NOE. Additionally, we report a closer direct association between two LINE-1s in different EPHA3 introns. Finally, hypermethylation of some LINE-1s can be found sporadically in cancer. In conclusion, even though the global hypomethylation process that occurs in cancerous cells can generally deplete LINE-1 methylation levels, LINE-1 methylation can be influenced differentially depending on where the particular sequences are located in the genome.

Genomic alterations in nasopharyngeal carcinoma: loss of heterozygosity and Epstein-Barr virus infection

Nasopharyngeal carcinoma is a subset of head and neck squamous cell cancers with unique endemic distribution and aetiological co-factors. Epstein-Barr virus has been revealed to be an important aetiological factor for most nasopharyngeal carcinomas. Nevertheless, additional genetic alterations may be involved in their development and progression. The aim of this study was to determine the likely chromosomal locations of tumour-suppressor genes related to Epstein-Barr virus-associated nasopharyngeal carcinoma. Fifty-six microsatellite polymorphic markers located on every autosomal arm were used to estimate the incidence of loss of heterozygosity in 27 Epstein-Barr virus-associated nasopharyngeal carcinomas. High frequencies of allelic loss were observed on chromosome 3p (75.0%) and 9p (87.0%). Chromosome 9q, 11q, 13q and 14q displayed loss in over 50%, while chromosome 3q, 6p, 16q, 19q and 22q exhibited loss in 35-50%. Furthermore, several other chromosomal arms demonstrated allelic loss in 20-35%. Additionally, 1 of the 27 cases showed microsatellite instability at multiple loci. These findings provide evidence of multiple genetic alterations during cancer development and clues for further studies of tumour-suppressor genes in Epstein-Barr virus-associated nasopharyngeal carcinoma.

Global Gene Expression Profile of Nasopharyngeal Carcinoma by Laser Capture Microdissection and Complementary DNA Microarrays

A number of genetic and epigenetic changes underlying the development of nasopharyngeal carcinomas have recently been identified. However, there is still limited information on the nature of the genes and gene products whose aberrant expression and activity promote the malignant conversion of nasopharyngeal epithelium. Here, we have performed a genome-wide transcriptome analysis by probing cDNA microarrays with fluorescent-labeled amplified RNA derived from laser capture microdissected cells procured from normal nasopharyngeal epithelium and areas of metaplasia-dysplasia and carcinoma from EBV-associated nasopharyngeal carcinomas. This approach enabled the identification of genes differentially expressed in each cell population, as well as numerous genes whose expression can help explain the aggressive clinical nature of this tumor type. For example, genes indicating cell cycle aberrations (cyclin D2, cyclin B1, activator of S-phase kinase, and the cell cycle checkpoint kinase, CHK1) and invasive-metastatic potential (matrix metalloproteinase 11, v-Ral, and integrin β4) were highly expressed in tumor cells. In contrast, genes underexpressed in tumors included genes involved in apoptosis (B-cell CLL/lymphoma 6, secretory leukocyte protease inhibitor, and calpastatin), cell structure (keratin 7 and carcinoembryonic antigen-related cell adhesion molecule 6), and putative tumor suppressor genes (H-Ras-like suppressor 3, retinoic acid receptor responder 1, and growth arrested specific 8) among others. Gene expression patterns also suggested alterations in the Wnt/β-catenin and transforming growth factor β pathways in nasopharyngeal carcinoma. Thus, expression profiles indicate that aberrant expression of growth, survival, and invasion-promoting genes may contribute to the molecular pathogenesis of nasopharyngeal carcinoma. Ultimately, this approach may facilitate the identification of clinical useful markers of disease progression and novel potential therapeutic targets for nasopharyngeal carcinoma.

Hypomethylation of Intragenic LINE-1 Represses Transcription in Cancer Cells through AGO2

In human cancers, the methylation of long interspersed nuclear element -1 (LINE-1 or L1) retrotransposons is reduced. This occurs within the context of genome wide hypomethylation, and although it is common, its role is poorly understood. L1s are widely distributed both inside and outside of genes, intragenic and intergenic, respectively. Interestingly, the insertion of active full-length L1 sequences into host gene introns disrupts gene expression. Here, we evaluated if intragenic L1 hypomethylation influences their host gene expression in cancer. First, we extracted data from L1base (http://l1base.molgen.mpg.de), a database containing putatively active L1 insertions, and compared intragenic and intergenic L1 characters. We found that intragenic L1 sequences have been conserved across evolutionary time with respect to transcriptional activity and CpG dinucleotide sites for mammalian DNA methylation. Then, we compared regulated mRNA levels of cells from two different experiments available from Gene Expression Omnibus (GEO), a database repository of high throughput gene expression data, (http://www.ncbi.nlm.nih.gov/geo) by chi-square. The odds ratio of down-regulated genes between demethylated normal bronchial epithelium and lung cancer was high (p<1E−27; OR = 3.14; 95% CI = 2.54–3.88), suggesting cancer genome wide hypomethylation down-regulating gene expression. Comprehensive analysis between L1 locations and gene expression showed that expression of genes containing L1s had a significantly higher likelihood to be repressed in cancer and hypomethylated normal cells. In contrast, many mRNAs derived from genes containing L1s are elevated in Argonaute 2 (AGO2 or EIF2C2)-depleted cells. Hypomethylated L1s increase L1 mRNA levels. Finally, we found that AGO2 targets intronic L1 pre-mRNA complexes and represses cancer genes. These findings represent one of the mechanisms of cancer genome wide hypomethylation altering gene expression. Hypomethylated intragenic L1s are a nuclear siRNA mediated cis-regulatory element that can repress genes. This epigenetic regulation of retrotransposons likely influences many aspects of genomic biology.