Aberrant Methylation Patterns Research Articles

Aberrant Methylation in CpG Islands of p15 and p16 Tumor Suppressor Genes in Pancreatic Cancer Tissue

Objective: To detect the aberrant methylation patterns in the CpG islands of p16 and p15 tumor suppressor genes, and to analyze its correlation with pancreatic carcinogenesis and with clinicopathological characteristics of patients with pancreatic cancer (PC). Methods: The methylation-specific polymerase chain reaction (MSP) method was used to monitor methylation patterns in the CpG islands of p15 and p16 genes from 29 cases of PC and 3 cases of chronic pancreatitis (CP) paraffin-embedded tissue, as well as 2 cases of normal liver tissues and 12 cases of normal blood samples. Results: p15 and p16 genes were detected to show unmethylation patterns and no amplification using methylation-specific primers in control group. The aberrant methylation rates of p16 in carcinoma tissue and adjacent noncarcinoma tissue were 37.9% (11 of 29 cases) and 34.5% (10 of 29 cases) respectively. Of the 11 aberrant methylated samples, 5 showed complete methylation and 6 hemimethylation. The methylation rates of p15 gene in carcinoma tissue and adjacent noncarcinoma tissue were 27.5% (8/29) and 24.4% (7/29) respectively. Of the 8 aberrant methylated samples, 3 showed complete methylation and 5 hemimethylation. In 6 PC samples, aberrant methylation in CpG islands of both p15 and p16 genes existed simultaneously. The aberrant methylation patterns in CpG islands of p15 and p16 genes had no close correlation with the clinicopathological characteristics (age, sex, smoking, volume of primary tumor, differentiation, clinical stage and histological classification) of the patients with PC (P>0.05). Conclusion: The aberrant methylation in CpG islands of p15 and p16 genes could be regarded as an early molecular event in PC and had no close correlation with the clinicopathological characteristics of the patients with PC.

Cloning of the hamster p16 gene 5′ upstream region and its aberrant methylation patterns in pancreatic cancer

The hamster model of pancreatic carcinogenesis is useful for understanding the development of human pancreatic cancer. However, there is only a small amount of hamster genetic information available for analyzing the gene alterations in hamster pancreatic cancers. Here, we determined the nucleotide sequence of the 5′ upstream region of the hamster p16 gene using a suppression polymerase chain reaction method combined with gene-specific primers. Based on this sequence, we analyzed the methylation status of the 5′ region by bisulfite sequencing in three normal pancreatic tissues and five pancreatic duct adenocarcinomas (PDAs). All five PDAs were highly methylated in the 5′ upstream region and showed reduced expressions of the p16 gene, while the three normal samples were demethylated. The method described in this study is a highly effective and rapid technique for determining the 5′ upstream region, and is applicable to epigenetic studies of the methylation status of this region.

A population-epigenetic model to infer site-specific methylation rates from double-stranded DNA methylation patterns.

Cytosine methylation is an epigenetic mechanism in eukaryotes that is often associated with stable transcriptional silencing, such as in X-chromosome inactivation and genomic imprinting. Aberrant methylation patterns occur in several inherited human diseases and in many cancers. To understand how methylated and unmethylated states of cytosine residues are transmitted during DNA replication, we develop a population-epigenetic model of DNA methylation dynamics. The model is informed by our observation that de novo methylation can occur on the daughter strand while leaving the opposing cytosine unmethylated, as revealed by the patterns of methylation on the two complementary strands of individual DNA molecules. Under our model, we can infer site-specific rates of both maintenance and de novo methylation, values that determine the fidelity of methylation inheritance, from double-stranded methylation data. This approach can be used for populations of cells obtained from individuals without the need for cell culture. We use our method to infer cytosine methylation rates at several sites within the promoter of the human gene FMR1.

Methylation profiles of genes utilizing newly developed CpG island methylation microarray on colorectal cancer patients

Aberrant methylation of DNA has been shown to play an important role in a variety of human cancers, developmental disorders and aging. Hence, aberrant methylation patterns in genes can be a molecular marker for such conditions. Therefore, a reliable but uncomplicated method to detect DNA methylation is preferred, not merely for research purposes but for daily clinical practice. To achieve these aims, we have established a precise system to identify DNA methylation patterns based on an oligonucleotide microarray technology. Our microarray method has an advantage over conventional methods and is unique because it allows the precise measurement of the methylation patterns within a target region. Our simple signal detection system depends on using an avidin–biotinylated peroxidase complex and does not require an expensive laser scanner or hazardous radioisotope. In this study, we applied our technique to detect promoter methylation status of O6-methylguanine-DNA methyltransferase (MGMT) gene. Our easy-handling technology provided reproducible and precise measurement of methylated CpGs in MGMT promoter and, thus, our method may bring about a potential evolution in the handling of a variety of high-throughput DNA methylation analyses for clinical purposes.

219 METHYLATION STATUS OF A DIFFERENTIALLY METHYLATED REGION (DMR) WITHIN THE BOVINE Igf2 GENE IN PREIMPLANTATION EMBRYOS

Specific DNA regions within imprinted genes become differentially methylated on the maternal and paternal chromosomes during germ cell development. These DMRs play a crucial role in the regulation of imprinted gene expression. The murine insulin-like growth factor2 gene (Igf2) is imprinted and contains an intragenic DMR within the last exon. Recently it became known that the bovine Igf2 gene is also imprinted (Dindot et al. 2004 Biol. Reprod. 71, 470–478) where we have now identified an intragenic DMR in the last exon with the paternal allele being methylated. Aberrant methylation patterns within the bovine Igf2 gene could result in deregulated gene expression and could therefore be involved in the development of fetal abnormalities such as the large offspring syndrome (LOS) in cattle. We have studied the methylation status of 27 CG dinucleotides within this DMR in bovine pre-implantation embryos of different origin by bisulfite sequencing. DNA was isolated from expanded blastocysts collected in vivo and generated by in vitro fertilization (IVF), somatic nuclear transfer (NT), and parthenogenesis (PA). Additionally, DNA was obtained from fibroblasts derived from a female and a male adult animal and used as donor cells for NT and from zygotes and 4-cell embryos both produced by IVF. After PCR amplification of the bisulfite-treated DNA, PCR products were cloned and sequenced. Methylation percentages were calculated for each individual clone by division of the 27 CpGs with the number of methylated CpGs per sample. The methylation levels (%) from each sample were then used to obtain the global methylation levels of the analyzed region. Methylation decreased during the transition from the zygote (28.4% ± 3.8 SEM) to the 4-cell embryo (6.3% ± 2.2 SEM) indicating that the DMR is demethylated after fertilization. An increased methylation level was observed in expanded blastocysts (in vivo: 10.2% ± 1.2 SEM; IVF: 10.1% ± 0.7 SEM; female NT: 12.4% ± 1.4 SEM). Thus, remethylation starts before the blastocyst stage. The higher methylation level of male NT blastocysts (22% ± 1.9 SEM) in comparison to their in vivo and IVF counterparts could be due to an insufficient reprogramming of the donor cells after nuclear transfer. Female and male donor cells were both heavily methylated (77% ± 2.2 SEM; 72% ± 2.9 SEM, respectively). Parthenogenetic expanded blastocysts were less methylated (2.3% ± 1 SEM), probably due to their diploid maternal genome. Results show for the first time that the methylation status at this DMR is associated with the origin of the embryo. Analysis of methylation patterns in pre-implantation embryos could provide a diagnostic tool to unravel mechanisms involved in fetal malformations often observed after the use of in vitro fertilization and/or nuclear transfer.

222 METHYLATION OF THE 52-UPSTREAM REGION OF THE H19 GENE IN MOUSE SOMATIC CELL, GAMETES, WILD TYPE AND ANDROGENETIC ES CELLS

In mammals, several genes influenced by the phenomenon of genomic imprinting are critical during development. Recently, Kono et al. (2004 Nature 428, 860–864) reported the production of intact female mouse individuals that had only two haploid sets of maternal genome. They obtained these mice by combining a normal haploid maternal genome and a mutant haploid maternal genome with a 13 k base deletion in the H19 gene and its 5′-upstream region. This genomic combination resulted in the appropriate expression of the Igf2, H19, and other imprinted genes. In the mouse genome, there are four CTCF-binding sites in the 5′-upstream region of the H19 gene. The binding of CTCF to these binding sites regulates the expression of the Igf2 and H19 genes. The binding of CTCF to its binding sites is regulated by methylation of CpG sites in binding sites. In this study, as the first step to elucidate the role of the paternal genomic imprinting during development, we investigated the methylation of CpG sites in the 5′-upstream region of the H19 gene in mouse somatic cells, gametes, and two types of ES cells. Genomic DNA was isolated from BDF1 (C57BL/6N × DBA/2N) mouse's tail (male and female somatic tissue, mST and fST, respectively), spermatozoa (S), oocytes (O), and wild type and androgenetic embryonic stem cells (wtES and agES, respectively). The methylation of CpG sites was evaluated by using the bisulfite sequencing assay. There were 13 CpG sites and a CTCF-binding site in the region from −4413 to −3976 in the H19 gene relative to the transcription start site. The percentages of CpG sites in this region that were methylated were 88% (160/182), 79% (27/130), 93% (230/247), 8% (10/130), 77% (10/13) and 89% (314/351) for mST, fST, S, O, wtES, and agES, respectively. In the CTCF-binding site core motif (CCGCGTGGTGGCAG), the percentages of methylated CpG sites were 93% (26/28), 80% (16/20), 95% (36/38), 0% (0/20), 50% (1/2) and 96% (52/54) for mST, fST, S, O, wtES, and agES, respectively. The CpG sites in the sequence of agES were highly methylated similar to the finding in spermatozoa. However, an aberrant methylation pattern was observed in some clones of agES. From these results, it was concluded that the methylation of CpG sites in the genomic sequence of agES was well conserved and, therefore, agES is useful to elucidate the role of the paternal genomic imprinting during development. This work was supported by Wakayama Prefecture Collaboration of Regional Entities for the Advanced of Technological Excellence, Japan, and by a Grant-in-Aid for the 21st Century COE Program of the Japan MEXT.

The DNA methylation events in normal and cloned rabbit embryos

To study the DNA methylation events in normal and cloned rabbit embryos, we investigated the methylation status of a satellite sequence and the promoter region of a single-copy gene using bisulfite-sequencing technology. During normal rabbit embryo development, both sequences maintained hypermethylation status until the 8- to 16-cell stage when progressive demethylation took place. In cloned embryos, the single-copy gene promoter sequence was rapidly demethylated and precociously de novo methylated, while the satellite sequence maintained the donor-type methylation status in all examined stages. Our results indicate that unique sequences as well as satellite sequences may have aberrant methylation patterns in cloned embryos.

Can Selenium Prevent Colorectal Cancer? A Signpost From Epidemiology

The limited epidemiology published heretofore on associations between selenium and colorectal adenomas or cancer has been mixed, inconclusive, and based predominantly on small studies. The epidemiology studies have been about evenly split between those showing a statistically significant (1–5) or suggestive (6,7) protective association and those showing a null (8–11) or suggestive harmful (12,13) association between selenium and colorectal adenomas or cancer. The strongest (and only clinical) previous support for a possible protective effect of selenium intake came from a large, randomized controlled trial of selenium in preventing nonmelanoma skin cancer (14). This trial showed a statistically significant reduction of colorectal cancer as a secondary endpoint (relative risk [RR] 0.58, 95% confidence interval [CI] 0.18 to 0.95) that attenuated after longer follow-up (RR 0.45, 95% CI 0.19 to 1.08) (15). A marginally statistically significant overall reduction in colorectal adenomas, which became stronger in association with the lowest tertile of baseline selenium, was also observed again, as a secondary endpoint in the trial (16). In this issue of the Journal, Jacobs et al. report their pooled analysis of three randomized clinical trials of dietary or nutrient interventions in preventing colorectal adenomas (17). Individuallevel data from each of the Wheat Bran Fiber (WBF) Trial (18), the Polyp Prevention Trial (PPT) (19), and the Polyp Prevention Study (PPS) (20) were statistically reanalyzed to provide a more precise estimate of the association between plasma selenium concentration and adenoma risk than would be possible from any of the three studies alone. None of the trials tested selenium as an intervention. This pooled analysis, the largest epidemiologic investigation of the role of selenium in preventing colorectal neoplasia, indicates that individuals in the highest quartile of plasma selenium concentration (median 150 ng/mL) (plasma collected at enrollment for PPT and PPS and 1 year after randomization for WBF) had 34% lower odds (OR 0.66, 95% CI 0.50 to 0.87, Ptrend .006) of developing a new adenoma throughout the follow-up, compared with those in the lowest quartile of plasma selenium (median 113 ng/mL). Plasma selenium concentrations were not statistically significantly associated with adenoma size, location, or histology in the pooled analysis. There was a trend, however, of fewer advanced adenomas ( 1 cm in diameter and/or villous histology) in persons within the highest quartile of plasma selenium. This suggestion of activity in higher–cancer risk, advanced adenomas supports the promise of selenium for reducing colorectal cancer risk. The association of selenium with colorectal cancer development (and mortality) will be assessed as a prespecified secondary endpoint of the Selenium and Vitamin E [prostate] Cancer Prevention Trial (SELECT) currently under way in more than 35 000 men (21). Although the three source studies were mutually independent and executed prior to the pooling project, they had similar designs and outcomes that strengthened the pooled analysis. All participants were at high adenoma risk after recent colonoscopic adenoma resection; each of the three trials assessed colorectal adenoma endpoints in over 85% of its patients, at year 1 and either year 3 (WBF) or year 4 (PPT and PPS) after randomization; and each intervention did not statistically significantly alter the adenoma risk. The pooled analysis also had limitations, however, which were adequately discussed by the authors and included the possibility that some events (new adenomas) detected at year 1 were not actual events but preexisting adenomas missed at the index colonoscopy, varied blood collection times, and different methodologies used to determine plasma selenium concentrations. The biologic plausibility of the pooled analysis and earlier secondary clinical data is provided by consistent animal studies showing the activity of selenium in colorectal neoplasia (22–28) and by other data that address relevant selenium mechanisms. Selenium has effects on key cellular events of tumorigenesis, such as cell proliferation and apoptosis, and the possible complex mechanisms underlying these effects are emerging in increasing numbers (28–30). Important relevant selenium mechanisms may involve gene promoter methylation and polyunsaturated fatty acid metabolism. Cancer cells commonly have aberrant methylation patterns characterized by global hypomethylation coupled with hypermethylation of CpG islands that appear to contribute importantly to tumorigenesis (31). The enzyme DNA cytosine methyltransferase 1 (DNMT1) is increased in tumor progression in association with regional hypermethylation (31). Preclinical studies suggest that selenium inhibits DNMT1 in various cell lines, including the HCT116 and HT-29 human colonic carcinoma cell lines (22,32–34). Oxidative metabolism of the n-6 polyunsaturated fatty acids arachidonic and linoleic acids contributes importantly to colonic tumorigenesis (35), and current data suggest that selenium and arachidonic and linoleic acid metabolism are linked in colonic tumorigenesis. Selenomethionine inhibited growth in four hu-

Molecular Biomarker in Prostate Cancer: The Role of CpG Island Hypermethylation

CpG island hypermethylation may be one of the earliest somatic genome alterations to occur during the development of multiple cancers. Recently, aberrant methylation patterns for different tumors have been reported. We present a comprehensive review of the literature describing the role of CpG island hypermethylation of DNA from prostatic tissue and bodily fluids from men with prostate cancer. We reviewed the literature to evaluate CpG island hypermethylation in tissue and bodily fluids of men with primary and metastatic prostate cancer. Additionally, we reviewed the literature with respect to CpG island hypermethylation patterns in other urological malignancies. Using modern analytic methods, CpG island hypermethylation detection can be achieved. In men with prostate cancer, correlations between specific gene regulatory region hypermethylation analyses and Gleason score, pathologic stage and tumor recurrence have been demonstrated. CpG island hypermethylation may serve as a useful molecular biomarker for the detection and diagnosis of patients with prostate cancer.

The ubiquitin-conjugating DNA repair enzyme HR6A is a maternal factor essential for early embryonic development in mice.

The Saccharomyces cerevisiae RAD6 protein is required for a surprising diversity of cellular processes, including sporulation and replicational damage bypass of DNA lesions. In mammals, two RAD6-related genes, HR6A and HR6B, encode highly homologous proteins. Here, we describe the phenotype of cells and mice deficient for the mHR6A gene. Just like mHR6B knockout mouse embryonic fibroblasts, mHR6A-deficient cells appear to have normal DNA damage resistance properties, but mHR6A knockout male and female mice display a small decrease in body weight. The necessity for at least one functional mHR6A (X-chromosomal) or mHR6B (autosomal) allele in all somatic cell types is supported by the fact that neither animals lacking both proteins nor females with only one intact mHR6A allele are viable. In striking contrast to mHR6B knockout males, which show a severe spermatogenic defect, mHR6A knockout males are normally fertile. However, mHR6A knockout females fail to produce offspring despite a normal ovarian histology and ovulation. The absence of mHR6A in oocytes prevents development beyond the embryonic two-cell stage but does not result in an aberrant methylation pattern of histone H3 at this early stage of mouse embryonic development. These observations support redundant but dose-dependent roles for HR6A and HR6B in somatic cell types and germ line cells in mammals.

Epigenetic changes in breast cancer shown by aberrant promoter methylation

The aberrant methylation of cytosine residues in the promoter region of growth-regulatory genes is now recognized as an important inactivation mechanism in addition to deletion and mutation in the development and progression of cancer. Principally all pathways regulating proliferation and differentiation, which are perturbed in cancer cells, can be affected. In breast cancer epigenetic inactivation is a frequent and early event displaying great quantitative gene-specific differences during progression. The occurrence of specific aberrant methylation patterns is associated with the histological grade and subtype of mammary carcinoma biopsies.

DLC-1 operates as a tumor suppressor gene in human non-small cell lung carcinomas

The deleted in liver cancer (DLC-1) gene at chromosome 8p21-22 is altered mainly by genomic deletion or aberrant promoter methylation in a large number of human cancers such as breast, liver, colon and prostate and is known to have an inhibitory effect on breast and liver tumor cell growth. Given the high frequency of deletion involving region 8p21-22 in human non-small cell lung carcinoma (NSCLC), we examined alterations of DLC-1 in a series of primary tumors and tumor cell lines and tested effects of DLC-1 on tumor cell growth. A significant decrease or absence of the DLC-1 mRNA expression was found in 95% of primary NSCLC (20/21) and 58% of NSCLC cell lines (11/19). Transcriptional silencing of DLC-1 was primarily associated with aberrant DNA methylation, rather than genomic deletion as 5-aza-2'-deoxycytidine induced reactivation of DLC-1 expression in 82% (9/11) NSCLC cell lines showing downregulated DLC-1. It was further evidenced by an aberrant DLC-1 promoter methylation pattern, which was detected by Southern blotting in 73% (8/11) of NSCLC cell lines with downregulation of the gene. The transfer of DLC-1 into three DLC-1 negative cell lines caused a significant inhibition in cell proliferation and/or a decrease in colony formation. Furthermore, stable transfer of DLC-1 abolished tumorigenicity in nude mice of two cell lines, suggesting that DLC-1 plays a role in NSCLC by acting as a bona fide new tumor suppressor gene.

Atypical methylation of the interleukin-8 gene correlates strongly with the metastatic potential of breast carcinoma cells.

Previously, we have shown that a strong correlation exists between the metastatic potential of breast carcinoma cell lines and their ectopic expression of IL-8. The undifferentiated, highly metastatic cell lines with high metastatic potential produce much more IL-8 than their differentiated lower metastatic counterparts. After eliminating the possibility that transcription factor activity was responsible for differences in IL-8 release, we examined the IL-8 gene for possible epigenetic modifications. Here, we report an aberrant methylation pattern that may be responsible for the differences in IL-8 release between the high and low metastatic cell lines. We determined that none of the deoxycytidylate-phosphate-deoxyguanylate (CpG) sites in the reported IL-8 promoter were methylated in either cell type. Much further upstream in the IL-8 gene, two CpG sites were identified that are differentially methylated. These two sites were fully methylated in the high metastatic cell lines, which produce large quantities of IL-8 and remain unmethylated in the low metastatic cell lines where the IL-8 gene is relatively silent. The DNA methylation results presented here differ from the common epigenetic paradigm in which methylation of promoter CpG islands silences gene expression, suggesting that there are additional epigenetic control mechanisms that as yet have not been fully appreciated or explored.

Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins.

Cytosine-5 DNA methylation is a critical signal defining heritable epigenetic states of transcription. As aberrant methylation patterns often accompany disease states, the ability to target cytosine methylation to preselected regions could prove valuable in re-establishing proper gene regulation. We employ the strategy of targeted gene methylation in yeast, which has a naturally unmethylated genome, selectively directing de novo DNA methylation via the fusion of C5 DNA methyltransferases to heterologous DNA-binding proteins. The zinc-finger proteins Zif268 and Zip53 can target DNA methylation by M.CviPI or M.SssI 5-52 nt from single zinc-factor binding sites. Modification at specific GC (M.CviPI) or CG (M.SssI) sites is enhanced as much as 20-fold compared with strains expressing either the free enzyme or a fusion protein with the zinc-finger protein moiety unable to bind to DNA. Interestingly, methylation is also selectively targeted as far as 353 nt from the zinc-finger protein binding sites, possibly indicative of looping, nucleosomes or higher-order chromatin structure. These data demonstrate that methylation can be targeted in vivo to a potentially broad range of sequences using specifically engineered zinc-finger proteins. Further more, the selective targeting of methylation by zinc-finger proteins demonstrates that binding of distinct classes of factors can be monitored in living cells.

DNA demethylating agents and chromatin-remodelling drugs: which, how and why?

DNA hypermethylation at the CpG dinucleotides clustered in "islands" in the promoter regions of genes causes transcriptional repression through the remodelling of chromatin. Aberrant methylation patterns of tumor suppressor genes and their subsequent silencing constitute a common feature of many cancers. Thus, the search for drugs that interfere in methylation-mediated gene repression has become one of the major goals in the design of cancer therapies. The major actors in the mammalian methylation system are DNA-methyltransferases (DNMTs), and methyl-CpG-binding proteins (MBDs), which recognize methylated cytosine and recruit repressor complexes, including histone deacetylases (HDACs). In this context, two major groups of drugs can be distinguished. The first one is constituted by substances that inhibit the action of DNMTs, either competing with cytosine or with S-adenosylmethionine (SAM, AdoMet) or acting over the DNMTs themselves. The second group involves compounds that inhibit subunits of the repressor complexes, such as HDACs. In this manuscript we review these two different groups of drugs, discussing their properties and the side effects that have been described (that occur by interference with other metabolic pathways). We also propose the logical pharmacological extension of these findings to design more specific and effective drugs for the prevention and treatment of human cancer.

Aberrant methylation patterns at the two-cell stage as an indicator of early developmental failure.

The fertilized mouse egg actively demethylates the paternal genome within a few hours after fertilization, whereas the maternal genome is only passively demethylated by a replication-dependent mechanism after the two-cell stage. This evolutionarily conserved assymetry in the early diploid mammalian embryo may have a role in methylation reprogramming of the two very different sets of sperm and egg chromatin for somatic development and formation of totipotent cells. Immunofluorescence staining with an antibody against 5-methylcytosine (MeC) showed that the incidence of abnormal methylation patterns differs between mouse two-cell embryos from superovulated females, nonsuperovulated matings, and in vitro fertilization (IVF). It also depends on embryo culture conditions and genetic background. In general, there was a good correlation with the number of embryos (from the same experiment) which did not develop in vitro up to the blastocyst stage. Thus, aberrant genome-wide DNA methylation in early embryos may be an important mechanism contributing to the high incidence of developmental failure in mammals. Similar to the situation in abnormally methylated embryos from nuclear transfer, it may cause a high incidence of pregnancy loss and abnormal phenotypes.

Restriction landmark genomic scanning for DNA methylation in cancer: past, present, and future applications

The field of molecular biology was revolutionized by the advent of gel electrophoresis. Restriction landmark genomic scanning (RLGS) is a type of two-dimensional electrophoresis employed in the genome-wide assessment of genomic alterations. RLGS has been used to study genetic and epigenetic changes in normal tissues, primary tumors, cancer cell lines, and various organisms such as mice, rats, hamsters, bacteria, and plants. An RLGS profile displays over 2000 radiolabeled restriction landmark sites in a single assay. When conducted with methylation-sensitive restriction enzymes whose sites are preferentially located in CpG island regulatory regions, RLGS becomes a very versatile tool for the investigation of both normal and aberrant methylation patterns. Early studies performed on tumor DNA were mainly descriptive in nature, essentially a catalogue of loci that were changed to varying degrees in different tumor types. Over time, as investigators have become more proficient with RLGS and have undertaken high-throughput studies, the need for efficient cloning, imaging, and analysis systems has become paramount. Current studies focus on identifying specific genes and pathways involved in deregulated methylation in cancer. As such, RLGS analysis of tumor samples has made tremendous contributions to our understanding of the role of DNA methylation in cancer. Future directions will take advantage of the abundant genomic sequence data available to link all of the RLGS loci to genes and create biologically relevant methylation profiles of cancer. This review discusses practical considerations of using RLGS as a genome scanning tool and the past, present, and future applications in cancer biology.

The impact of chromatin in human cancer: linking DNA methylation to gene silencing.

For decades, chromatin was considered to be an inert structure whose only role was the compacting and confining of DNA inside the eukaryotic nucleus. However, tremendous progress in this field over the last 10 years has dramatically elevated chromatin to a key position in the control of gene activity. Its role in mediating the transformation of a normal cell into a malignant state is particularly interesting. On one side of this story there is the discovery that aberrant methylation patterns in an increasing number of tumour suppressor and DNA repair genes determine carcinogenetic transformation; while on the other side, there is the existence of a series of methyl-DNA binding activities that recruit co-repressor complexes and modify the structure of the chromatin to produce a transcriptionally silenced state. Although this field has seen rapid progress in recent years, detailed mechanisms by which this machinery modifies chromatin structure to its appropriate state and the specific targeting of repressor complexes have yet to be resolved. In this review we present the models of how repressor complexes may modify chromatin structure and mediate silencing of tumour suppressor and DNA repair genes.

Analysis of 11q21-24 loss of heterozygosity candidate target genes in breast cancer: indications of TSLC1 promoter hypermethylation.

Loss of heterozygosity (LOH) at the distal half of chromosome arm 11q is frequent in a variety of human tumors, including breast cancer, and is often associated with poor prognosis. In an ongoing attempt to locate and characterize the main target genes within this chromosome region, we first looked for aberrations in known genes either suggested to be involved in tumorigenesis or shown to suppress tumor formation. We examined 31 primary breast tumors showing LOH in 11q21-24 for mutations in the MRE11A, CHK1, PPP2R1B, and TSLC1 genes. The absence of intragenic alterations related to cancer led us next to evaluate possible gene silencing resulting from promoter region CpG hypermethylation, using the bisulfite sequencing technique. In addition to the four genes mentioned above, we also analyzed the ATM gene, which had been investigated for certain germline mutations in an earlier study. Only the TSLC1 promoter region exhibited aberrant methylation patterns, and altogether 33% (10/30) of the successfully analyzed tumors showed evidence of elevated levels of TSLC1 CpG methylation. Ten percent (3/30) of the tumors showed significantly increased methylation. Thus, as has been shown in lung and some other forms of cancer, hypermethylation of the TSLC1 promoter region is also frequently a second hit along with LOH in breast cancer.

Typical Demethylation Events in Cloned Pig Embryos: CLUES ON SPECIES-SPECIFIC DIFFERENCES IN EPIGENETIC REPROGRAMMING OF A CLONED DONOR GENOME

We investigated whether a genomic demethylation process occurs in pig preimplantation embryos produced by either normal fertilization or somatic cell nuclear transfer. The methylation status of the centromeric satellite and the PRE-1 short interspersed element (SINE) sequences was characterized using bisulfite-sequencing technology. Typical demethylation processes were identified in these repetitive sequences of the cloned donor genome during cleavage, the patterns of which were similar to the ones detected in fertilized counterparts. These findings are different from previous observations with cloned bovine embryos where various repeated regions of the donor genome exhibited aberrant methylation patterns. Our results indicate that species-specific differences exist in modifying the epigenetic status of cloned donor genomes.