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Dissecting Complex Epigenetic Alterations in Breast Cancer Using CpG Island Microarrays
Abstract
It is now clear that aberrant DNA methylation observed in cancer cells is not restricted to a few CpG islands, but affects multiple loci. When this epigenetic event occurs at the 5'-end of the regulatory region of genes, it is frequently associated with transcriptional silencing. To investigate further this widespread event in the tumor genome, we developed a novel microarray containing 7776 short GC-rich tags tethered to glass slide surfaces. This DNA chip was used to study 17 paired tissues of breast tumors and normal controls. Amplicons, representing differential pools of methylated DNA fragments between tumors and normal controls, were cohybridized to the microarray panel. Hypermethylation of multiple CpG island loci was then detected in a two-color fluorescence system. Approximately 1% (on average, 83 loci) of these CpG islands examined were hypermethylated in this patient group. Hierarchical clustering segregated these tumors based on their methylation profiles and identified a group of CpG island loci that corresponds to the hormone-receptor status of breast cancer. This observation was independently confirmed by examining a single locus, the promoter of the human glypican 3 gene, which was predominately hypermethylated in the hormone receptor-negative tumors. Our findings support the notion that hypermethylation of critical CpG island loci influences cancer development and produces distinct epigenetic signatures for particular tumor subtypes.
... Such a finding has neither been determined nor reported in other studies examining the GPC3 protein expression in breast cancer subtypes. Our results are also consistent with previous studies showing weak or absent GPC3 protein expression in other breast cancer subtypes [13][14][15][16]28]. Baumhoer et al. reported that GPC3 expression was weakly identified only in 17% of medullary breast carcinomas, 20% of lobular breast carcinomas, and 15% of mucinous carcinomas [15]. ...
... Moreover, similar results were reported by two other studies demonstrating GPC3 expression in 13% and 17% of breast cancer samples [13,16]. A possible explanation for these results came from two studies reporting the absence or weak expression of GPC3 in certain breast cancer subtypes due to the silencing of GPC3 by GPC3 promoter hypermethylation [14,28]. This downregulation of GPC3 was more predominant in patients who were negative for hormone receptors [28]. ...
... A possible explanation for these results came from two studies reporting the absence or weak expression of GPC3 in certain breast cancer subtypes due to the silencing of GPC3 by GPC3 promoter hypermethylation [14,28]. This downregulation of GPC3 was more predominant in patients who were negative for hormone receptors [28]. Such a finding was evident in the heterogeneity of GPC3 expression among different and certain breast cancer histological subtypes [13,15,16]. ...
Background and Objectives: breast cancer remains the most common health burden affecting females worldwide. Despite developments in breast cancer diagnostic approaches and treatment strategies, the clinical management of metastatic breast cancer remains challenging. Thus, there is a need to identify new biomarkers and novel drug targets for breast cancer diagnosis and therapy. Recently, aberrant glypican-3 (GPC3) expression in cancers has gained considerable interest in cancer research. The studies, however, have yielded contradictory results about GPC3 expression in breast cancer. Therefore, the current study aims to analyse GPC3 expression across a large panel of different breast cancer subtypes. Materials and Methods: GPC3 expression was immunohistochemically evaluated in 230 breast cancer patients along with eight normal tissues and its associations to clinical and demographic characteristics, as well as immunohistochemical biomarkers for breast cancer. Moreover, a public database consisting of breast cancer patients’ survival data and GPC3 gene expression information was used to assess the prognostic value of GPC3 in the survival of breast cancer patients. Results: GPC3 expression was only characterised in 7.5% of different histological breast cancer subtypes. None of the normal breast tissues displayed GPC3 expression. Interestingly, all cases of Paget’s disease, as well as 42.9% of intraductal and 16.7% of mucinous carcinomas were found to have GPC3 expression, where it was able to significantly discriminate Paget’s disease and intraductal carcinoma from other breast cancer subtypes. Importantly, GPC3 expression was found more often in tumours that tested positive for the expression of hormone receptors and human epidermal growth factor receptor 2 (HER2), indicating more favourable histological subtypes of breast cancer. Consequently, longer relapse-free survival (RFS) was significantly correlated with higher GPC3 mRNA expression. Conclusion: Our study proposes that GPC3 is a promising breast cancer subtype-specific biomarker. Moreover, GPC3 may have the potential to be a molecular target for the development of new therapeutics for specific subtypes of breast cancer.
... The current self-enforcing model implies close correlation of histone modifications and DNA methylation, especially the crosstalk between methyl-H3K9 and DNA methylation [82]. However, recent reports demonstrated that these epigenetic markers have varying degrees of autonomy [142,[269][270][271][272][273][274][275][276]. For example in Arabidopsis, Trichostatin A (TSA), a histone deacetylase inhibitor, and 5'-aza-2-deoxycytidine (AzadC), a demethylating agent, do not always produce redundant outcomes. ...
... were purchased from Upstate Biotechnology (Charlottesville, VA) and Abcam Pearson's correlation analysis (from 0.77-0.82) [275]. After excluding the spots flagged for bad quality, normalized Cy5/Cy3 ratios of these loci were calculated by GenePix Pro 6.0 [183]. ...
... Differential methylation hybridization (DMH) was performed essentially as described ( [183,275]). Briefly, 2 μg of genomic DNA were digested by MseI to produce small fragments and then H-24/H-12 PCR linkers (5' -AGGCAACTGTGCTATCCGAGGGA T-3' and 5'-TAATCCCTCG-GA-3') were ligated to the digested DNA fragments. ...
... Accumulating evidence indicates that this type of epigenetic alteration can be one of the most prevalent molecular markers for human cancers, and the list of abnormally methylated genes in various tumor types continues to grow (Jones and Baylin, 2002). To dissect complex epigenetic patterns in cancer, we developed a microarray-based approach in which thousands of short CpG island tags known as probes were arrayed on glass slides (Yan et al., 2001). DNA targets representing different pools of methylated CpG islands in tumor or control samples were used to hybridize the microarray panel. ...
... DNA targets representing different pools of methylated CpG islands in tumor or control samples were used to hybridize the microarray panel. This microarray approach was used to assess aberrant DNA methylation profiles in breast, ovarian, colorectal and endometrial carcinomas, and gliomas (Yan et al., 2001;Wei et al., 2002). Clustering analysis identified tumor subtypes that were associated with various clinicopathological parameters, including hormone receptor status, age at diagnosis, and the duration of patients' survival. ...
... The positive effect on survival correlates with findings from a number of authors who found that GPC3 has a protective role against human breast cancer progression and is in turn downregulated in breast cancer (Fernández-Vega et al. 2013). Two studies have shown that in breast cancer, GPC3 is downregulated by means of hypermethylation of the GPC3-promoter (Xiang et al. 2001;Yan et al. 2001) and Yan et al. have described that the silencing of GPC3 by means of promoter hypermethylation is more predominant in hormone-receptor negative patients, implying that GPC3 is expressed less in those patients. ...
Purpose
Dysregulated expression of proteoglycans influences the outcome and progression of numerous cancers. Several studies have investigated the role of individual glypicans in cancer, however, the impact of the whole glypican family of heparan sulfate proteoglycans on prognosis of a large patient cohort of breast cancer patients has not yet been investigated. In the present study, our aim was to investigate the prognostic power of the glypicans in breast cancer patients.
Methods
We used a public database including both gene expression data and survival information for 3951 breast cancer patients to determine the prognostic value of glypicans on relapse-free survival using Cox regression analysis. Moreover, we performed quantitative Real-Time PCR to determine glypican gene expression levels in seven representative breast cancer cell lines.
Results
We found that high GPC3 levels were associated with a better prognosis in overall breast cancer patients. When stratified by hormone receptor status, we found that in worse prognosis subtypes low GPC1 levels correlate with a longer relapse-free survival, and in more favorable subtypes low GPC6 was associated with longer survival.
Conclusion
Our study concludes that glypicans could act as subtype-specific biomarkers for the prognosis of breast cancer patients and sparks hope for future research on glypicans possibly eventually providing targets for the treatment of the disease.
... This gives a set of parallel DNA pools amplified and labeled with different fluorescent dyes. The relative fluorescent intensities provide information about DNA methylation patterns (Yan et al., 2001(Yan et al., , 2009). However, this method is not much sensitive and can't provide nucleotide-specific DNA methylation information, and is limited to eukaryotic epigenetic analysis or cytosine-methylation in prokaryotes only. ...
Human gut harbors diverse microorganisms influencing host metabolism and homeostasis. But evidence about the epigenetic mechanisms influencing gut microbiome as well as host is also known. Factors like diet, exercise, and age influence the gut microbiota. Microbial-derived metabolites decide the species richness of microbiome and induce specific epigenetic marks which have an impact on health and diseased conditions of host. Techniques like array hybridization, chromatin immunoprecipitation, and sequencing help to map the epigenetic changes. This article provides an overview of existing evidence toward interplay between epigenetic regulations of host-gut microbiome and highlights the future prospects of host-gut microbiome-epigenetic axis.
... genome is constitutively hypermethylated at Satellite I sequence (high copy number) and 5S rRNA genes of the oocyte in comparison to genes with a single-copy number that expresses a locus-specific methylation status [150]. Restriction enzymes have been used in combination with microarrays in the form of CpGs island microarrays to establish genome-wide DNA methylation profiles in human breast cancer cell lines [151], lung cancer, colon cancer and pancreatic cancer cell lines [152]. Also, direct sequencing is being combined with restriction enzymes to demonstrate global DNA methylation patterns in the human brain [153]. ...
Somatic cell nuclear transfer (SCNT) technique has been proving its worth for more than two decades now as over 20 different species have been successfully cloned. SCNT protocol for cloning is well established but efficiency in terms of live birth rate is still low. Epigenetic abnormality following nuclear reprogramming is considered as the main culprit behind its low efficiency. DNA methylation is one of the most important epigenetic modifications that directly or indirectly regulate gene expression pattern, development and genome stability. Embryos produced through SCNT are found to express abnormal DNA methylation profile in comparison with in vivo or in vitro produced embryos. In order to improve DNA methylation profile in cloned embryos, a complete database of whole genome is required to find out specific faulty targets. Many techniques including low throughput and high throughput approach has been used to profile DNA methylation pattern in bovine embryos throughout the developmental stages. In the present review, we have compiled the overall status of global DNA methylation, the effect of aberrant DNA methylation on development and evolution in methodologies used for profiling global DNA methylome in cloned embryos.
... The revolution of functional genomics truly benefited from the adaptation of microarray hybridization techniques [146][147][148][149]. The development of array hybridization began in the middle 1990 s. Subsequently, advances and implementation of this technology greatly facilitated DNA methylomic analysis, which has a much higher resolution than gel-based profiling [150][151][152]. ...
DNA methylation provides a pivotal layer of epigenetic regulation in eukaryotes that has significant involvement for numerous biological processes in health and disease. The function of methylation of cytosine bases in DNA was originally proposed as a "silencing" epigenetic marker and focused on promoter regions of genes for decades. Improved technologies and accumulating studies have been extending our understanding of the roles of DNA methylation to various genomic contexts including gene bodies, repeat sequences and transcriptional start sites. The demand for comprehensively describing DNA methylation patterns spawns a diversity of DNA methylation profiling technologies that target its genomic distribution. These approaches have enabled the measurement of cytosine methylation from specific loci at restricted regions to single-base-pair resolution on a genome-scale level. In this review, we discuss the different DNA methylation analysis technologies primarily based on the initial treatments of DNA samples: bisulfite conversion, endonuclease digestion and affinity enrichment, involving methodology evolution, principles, applications, and their relative merits. This review may offer referable information for the selection of various platforms for genome-wide analysis of DNA methylation.
... Affinity enrichment methods are based on the initial isolation of CpG islands from genomic DNA using a methyl-binding protein. The methylation status of the isolated region is confirmed by HpaII enzyme digestion and the sequence is detected by Southern blotting or microarray-based analysis [41]. For example, Weber et al. developed methylated DNA immunoprecipitation (MeDIP) [27], which uses an antibody specific to the 5-methylcytosine to isolate the methylated regions from fragmented DNAs. ...
Eukaryotic cell DNA conserves a distinct genomic methylation pattern, which acts as a molecular switch to control the transcriptional machinery of the cell. However, pathological processes can alter this methylation pattern, leading to the onset of diseases such as cancer. Recent advances in methylation analysis provide a more precise understanding of the consequence of DNA methylation changes towards cancer progression. Consequently, the discoveries of numerous methylation-based biomarkers have inspired the development of simple tests for cancer detection. In this opinion article, we systematically discuss the benefits and challenges associated with the promising methylation-based approaches and develop a point-of-care index to evaluate their potential in terms of point-of-care cancer diagnostics.
... Mass spectroscopy to high-performance liquid chromatography (HPLC)-based techniques have been applied to avoid chemical treatment in addition to gain quick and sensitive detection of DNA methylation (51). Other sophisticated detection techniques that have been reported are combined bisulfite restriction analysis (COBRA) (52), microarray-based DNA methylation profiling (53), surface plasmon resonance-based assays (54), etc. However, these methods are relatively expensive and require large volume of DNA. ...
... Le champ d'application des puces à ADN est vaste. Initialement développées comme outil de criblage de banque (197), il est possible grâce aux puces à ADN de mesurer l'expression des gènes (169,197), de décrire physiquement un génome (198), de définir le statut de méthylation de l'ADN (199,200), des histones (201), et d'identifier des intéractions ADN-protéines (202,203). Elles peuvent participer à l'annotation d'un génome (204) et à la détection des gènes (205). ...
Une des caractéristiques les plus connues de la programmation métabolique est qu’un événement commun physiopathologique à l'âge adulte obtenu indépendamment du stress nutritionnel au début de la vie. Cela a conduit à penser que les altérations métaboliques dûes au stress nutritionnel précoce pouvaient résulter de la programmation seulement d’un petit nombre de gènes qui agissent comme gardiens d'un réseau de gènes ou d'une voie de signalisation. Ici nous avons l’intention de tester cette hypothèse par l'analyse combinée du transcriptome et méthylome avec des échantillons de foie des rats nés de mères nourries avec une alimentation restreinte en protéines (MPR) ou carencée en donneur de méthyles (MDD) pendant la gestation et la lactation et comparer entre les 2 modèls. Au moment du sevrage, la progéniture MDD a été sacrifiée, tandis que la progéniture du groupe MPR a reçu une nourriture standard jusqu'à l'âge de 6 mois. Les rats à jours 21 nés de mères nourries avec un régime MDD ont 3.269 gènes surexprimé (P <0,0009) et 2.841 gènes sous-exprimés (P <0,0004) par rapport aux témoins. Les modifications de méthylation de l'ADN ont été trouvées dans les régions promotrices de 1.032 gènes. Les analyses fonctionnelles ont révélé que ces gènes sont principalement impliqués dans le métabolisme des lipides et du glucose, du système nerveux, la coagulation, le stress du réticulum endoplasmique et la fonction mitochondriale. Les master genes présentant des changements à la fois dans l'expression et la méthylation d'ADN sont limités à 266 gènes et ils sont principalement impliqués dans le système rénine-angiotensine, le métabolisme de la mitochondrie et de l'homéostasie phospholipide. La plupart de ces master genes participent à la Non Alcoholic Fatty Liver Disease (NAFLD). La restriction protéique maternelle (MPR) a entraîné une augmentation de la masse grasse abnominale, de l'hypertriglycéridémie, de l'hypercholestérolémie et un taux élevé d’acides gras par rapport aux témoins. 3.020 gènes sont surexprimés (P<0,0003) et 3.601 sous-exprimés (P<0,002) au niveau du transcriptome et 3.968 gènes modifiés au niveau du méthylome par rapport aux témoins. L'analyse fonctionnelle a indiqué que les gènes surexprimés sont principalement impliqués dans les voies métaboliques et les gènes sous-exprimés et différemment méthylés sont principalement impliqués dans des processus du développement. 998 master genes ont été trouvés, et léanalyse fonctionnelle de ces gènes a indiqué un effet significatif sur le développement des tissus, la régulation de la transcription et le métabolisme, et beaucoup d'entre eux sont associés à des maladies chroniques comme l'hypertension, l'obésité centrale et le diabète. L'expression des gènes et la méthylation de l'ADN du génome obtenus en utilisant ces modèles ont été comparés aux données de méthylome et de transcriptome précédemment obtenus à partir de foie des rats restreints en protéines et sacrifiés à la naissance. Cette analyse a révélé un ensemble commun de 46 gènes qui sont sur-exprimés et 42 gènes sous-exprimés dans les trois modèles de programmation métabolique par rapport aux animaux témoins. La plupart des gènes surexprimés sont impliqués dans la régulation de la fonction mitochondriale alors que les gènes sous-exprimés sont principalement impliquées dans la régulation de la prolifération cellulaire et l'expression des gènes. Nous avons identifié également un ensemble de 122 gènes dont les niveaux de méthylation ont été modifiés à la fois par une carence en donneurs de méthyle et une restriction protéique. Ces observations soutiennent l’hypothèse qu’un petit nombre de gènes essentiels sont à la base de la programmation de troubles métaboliques, indépendamment du stress nutritionnel
... Epigenetics & Chromatin *Correspondence: piferrer@icm.csic.es 1 Institut de Ciències del Mar, Consejo Superior de Investigaciones Científicas (CSIC), Passeig Marítim, [37][38][39][40][41][42][43][44][45]08003 Barcelona, Spain Full list of author information is available at the end of the article to control the expression of genes involved in the onset of cancer [10]. Furthermore, many tumour cells have hypermethylation in the promoters of tumour suppressor genes [11,12] and thus research has also contemplated the effects of demethylating agents to regain the expression of these silenced genes [13,14]. ...
Background:
The role of epigenetic modifications such as DNA methylation during vertebrate sexual development is far from being clear. Using the zebrafish model, we tested the effects of one of the most common DNA methyltransferase (dnmt) inhibitor, 5-aza-2'-deoxycytidine (5-aza-dC), which is approved for the treatment of acute myeloid leukaemia and is under active investigation for the treatment of solid tumours. Several dose-response experiments were carried out during two periods, including not only the very first days of development (0-6 days post-fertilization, dpf), as done in previous studies, but also, and as a novelty, the period of gonadal development (10-30 dpf).
Results:
Early treatment with 5-aza-dC altered embryonic development, delayed hatching and increased teratology and mortality, as expected. The most striking result, however, was an increase in the number of females, suggesting that alterations induced by 5-aza-dC treatment can affect sexual development as well. Results were confirmed when treatment coincided with gonadal development. In addition, we also found that the adult gonadal transcriptome of 5-aza-dC-exposed females included significant changes in the expression of key reproduction-related genes (e.g. cyp11a1, esr2b and figla), and that several pro-female-related pathways such as the Fanconi anaemia or the Wnt signalling pathways were downregulated. Furthermore, an overall inhibition of genes implicated in epigenetic regulatory mechanisms (e.g. dnmt1, dicer, cbx4) was also observed.
Conclusions:
Taken together, our results indicate that treatment with a DNA methylation inhibitor can also alter the sexual development in zebrafish, with permanent alterations of the adult gonadal transcriptome, at least in females. Our results show the importance of DNA methylation for proper control of sexual development, open new avenues for the potential control of sex ratios in fish (aquaculture, population control) and call attention to possibly hidden long-term effects of dnmt therapy when used, for example, in the treatment of prepuberal children affected by some types of cancer.
... Techniques for gene-specific DNA methylation analysis include methylation-sensitive restriction enzyme-Southern (MSRE-Southern) (48), methylation-sensitive restriction endonuclease-PCR (49,50), methylation-specific PCR (51), methylation-sensitive single-nucleotide primer extension (52), direct bisulfite genomic sequencing (49), methylight PCR (53,54), methylation-specific single-strand conformation analysis (55)(56)(57), methylation-specific denaturing gradient gel electrophoresis (58), methylation-specific melting curve analysis (59), methylation-specific denaturing highperformance liquid chromatography (60), and methylationspecific oligonucleotide microarray (61). Methylation pattern/profiling analysis includes restriction landmark genomic scanning (62), methylated CpG island amplification (63)-representational difference analysis (64) methylation-sensitive arbitrarily primed PCR (65), and differential methylation hybridization (66,67). ...
Hepatocellular carcinoma (HCC) is a common malignant tumor with high incidence and morbidity worldwide. The pathogenesis and progression of HCC are closely related to abnormal epigenetic regulation of hepatocytes. DNA methylation is an important regulatory mechanism in epigenetics which has been deeply and widely investigated. Along with the development of high-throughput sequencing techniques, including high-throughput gene chip analysis and next generation sequencing, great progress was achieved toward the understanding of the epigenetic regulation of HCC. In this article we will systematically summarize the current and emerging research on DNA methylation in HCC.
... The protocol of differential methylation hybridization (DMH) on human CpG island microarray was based on our previously described [23,24]. Briefly, the purified methylation amplicons (5 μg) from eight pairs of gastric tumor and non-tumor obtained from the tissue bank of Taichung Veterans General Hospital, Taiwan, were conducted using the BioPrime DNA labeling system (Invitrogen, CA, USA). ...
Introduction:
Regulator of chromosome condensation 1 (RCC1) is a critical cell cycle regulator. We firstly identified RCC1 gene hypermethylation in gastric tumor tissues using the differential methylation hybridization (DMH) microarray, but the role of RCC1 in the pathogenesis of gastric carcinoma is largely unknown.
Methods:
Three gastric cancer cell lines (AGS, MKN45, and TSGH9201) were used to analyze RCC1 gene methylation, mRNA and protein expressions. Furthermore, 85 pairs of matched human gastric carcinoma samples in a tissue microarray were used to analyze RCC1 expression by immunohistochemistry staining.
Results:
A differential methylation pattern was found in TSGH9201 (100%), MKN45 (87%), and AGS (62%) cell lines at the 9th CpG site of RCC1 exon 1. RCC1 mRNA and protein expressions in AGS cells were significantly higher than in TSGH9201 and MKN45 cell lines (P < 0.05). Tissue array data showed that RCC1 expression was detected in 21% (18/85) of gastric carcinoma tissues and in 80% (76/95) of adjacent non-tumor tissues. The expression of RCC1 in gastric carcinoma tissues was significantly lower than in adjacent non-tumor tissues (P < 0.001). Furthermore, an association between RCC1 expression and clinicopathological features showed that RCC1 expression was closely correlated with tumor differentiation and depth of invasion (P < 0.05).
Conclusions:
Our data indicate that RCC1 expression is frequently lost in poorly differentiated gastric cell lines and gastric carcinoma tissues. Loss of RCC1 expression is correlated with tumor differentiation and depth of invasion. These findings suggest that RCC1 may play a tumor suppressor role in gastric carcinoma.
... CpG islands methylation in the promoter region is one of the main mechanisms of transcriptional silence of ESR1 gene [60,61]. To investigate whether promoter methylation play roles in inhibition of ESR1 expression at the transcriptional level by (C) MCF7 cells were exposed to neo-tanshinlactone (2 M) in the presence of protein synthesis inhibitor cycloheximide (CHX, 10 g/ml) for 24 h. ...
Breast cancer, the most frequent cancer in women, is the second leading cause of cancer-related death. Estrogens and estrogen receptors are well recognized to play predominant roles in breast cancer development and growth. Neo-tanshinlactone is a natural product isolated from Salvia miltiorrhiza and showed selective growth inhibition of ER+ breast cancer cell lines as demonstrated by cell proliferation assay and colony formation assay. The selective anti-proliferative effect of neo-tanshinlactone was associated with the induction of apoptosis in ER+ breast cancer cells. We also found that neo-tanshinlactone decreased steady state ESR1 mRNA levels in ER+ breast cancer cells, which was further confirmed by analysis of ER protein levels as well as the mRNA levels of target genes of this transcription factor, such as ESR2, BRCA1, CCND1, GREB1, TFF1, SERPINB9 and ABCA3. Furthermore, analysis of heterogeneous nuclear RNA (hnRNA) demonstrated that neo-tanshinlactone inhibited ESR1 mRNA de novo synthesis. The decrease of steady state ESR1 mRNA upon neo-tanshinlactone treatment was not abolished by protein synthesis inhibitor cycloheximide. And inhibition of mRNA synthesis with actinomycin D revealed no significant effect of neo-tanshinlactone on ESR1 mRNA stability. These results indicated that transcriptional down-regulation of ESR1 mRNA could contribute to the selective activity of neo-tanshinlactone on ER+ breast cancer cells. And as expected, the combination of neo-tanshinlactone and antiestrogen reagent tamoxifen showed a synergistic effect on growth of ER+ MCF7 cells. Our results suggest that neo-tanshinlactone is a promising regimen for ER+ breast tumors.
... GPC-3 expression is elevated in several cancer types, such as embryonic tumors [33,34] , malignant melanoma [35] , and, most notably, HCC [36,37] . On the other hand, it is downregulated in malignant mesothelioma and ovarian cancer [38,39] , and silenced via promoter hypermethylation in the majority of breast cancers [40,41] . The effects of GPC-3, such as the growth-inhibitory potential, have been associated with the negative regulation of Hedgehog signaling [42] . ...
Proteoglycans are a group of molecules that contain at least one glycosaminoglycan chain, such as a heparan, dermatan, chondroitin, or keratan sulfate, covalently attached to the protein core. These molecules are categorized based on their structure, localization, and function, and can be found in the extracellular matrix, on the cell surface, and in the cytoplasm. Cell-surface heparan sulfate proteoglycans, such as syndecans, are the primary type present in healthy liver tissue. However, deterioration of the liver results in overproduction of other proteoglycan types. The purpose of this article is to provide a current summary of the most relevant data implicating proteoglycans in the development and progression of human and experimental liver cancer. A review of our work and other studies in the literature indicate that deterioration of liver function is accompanied by an increase in the amount of chondroitin sulfate proteoglycans. The alteration of proteoglycan composition interferes with the physiologic function of the liver on several levels. This article details and discusses the roles of syndecan-1, glypicans, agrin, perlecan, collagen XVIII/ endostatin, endocan, serglycin, decorin, biglycan, asporin, fibromodulin, lumican, and versican in liver function. Specifically, glypicans, agrin, and versican play significant roles in the development of liver cancer. Conversely, the presence of decorin could potentially provide protective effects.
... Since DNA methylation plays an important role in numerous biological processes, more and more techniques such as MSAP, MS-RDA, bisulfite sequencing, and microarray analysis [20][21][22][23] are developed to analyze methylation patterns or to screen for genes regulated by methylation mechanism. However, MSAP may lead to loss of target genes because it is difficult to distinguish different sequences with the same length using these techniques. ...
Stress-induced ROS changes DNA methylation patterns. A protocol combining methylation-sensitive restriction endonuclease (MS-RE) digestion with suppression subtractive hybridization (SSH) to construct the differential-methylation subtractive library was developed for finding genes regulated by methylation mechanism under cold stress. The total efficiency of target fragment detection was 74.64%. DNA methylation analysis demonstrated the methylation status of target fragments changed after low temperature or DNA methyltransferase inhibitor treatment. Transcription level analysis indicated that demethylation of DNA promotes gene expression level. The results proved that our protocol was reliable and efficient to obtain gene fragments in differential-methylation status.
... Dysregulation of lncRNAs is associated with many human diseases, including various types of cancers [64]. Many studies have used lncRNA microarrays to demonstrate lncRNA gene expression profiles and the prognostic potential of lncRNA profiles in various cancers [65,66]. ...
DNA microarray technologies have advanced rapidly and had a profound impact on examining gene expression on a genomic scale in research. This review discusses the history and development of microarray and DNA chip devices, and specific microarrays are described along with their methods and applications. In particular, microarrays have detected many novel cancer-related genes by comparing cancer tissues and non-cancerous tissues in oncological research. Recently, new methods have been in development, such as the double-combination array and triple-combination array, which allow more effective analysis of gene expression and epigenetic changes. Analysis of gene expression alterations in precancerous regions compared with normal regions and array analysis in drug-resistance cancer tissues are also successfully performed. Compared with next-generation sequencing, a similar method of genome analysis, several important differences distinguish these techniques and their applications. Development of novel microarray technologies is expected to contribute to further cancer research.
... Several global methylation assays are under development, including a high-density array of CpG islands (Yan et al., 2001). However, the approach requires a tedious, somewhat tricky sample preparation, and the current array of about 8,000 CpG islands is a significant underrepresentation of the estimated 45,000 that may be in the genome (admittedly not all of which are upstream regulatory elements of transcribed genes) (Costello et al., 2000). ...
The draft human genome sequence and the dissemination of high throughput technology provides opportunities for systematic analysis of cancer cells. Genome-wide mutation screens, high resolution analysis of chromosomal abberations and expression profiling all give comprehensive views of genetic alterations in cancer cells. From these analyses will come a complete list of the genetic changes that drive malignant transformation and of the therapeutic targets that may be exploited for clinical benefit.
... Signal intensities for singlecopy sequences were normalized and loci having Cy5 to Cy3 ratio cut-off ratios ≥1.5 were scored as hypermethylated. This method was previously used to identify hypermethylated loci in several tumor types (19). Data were then exported and subjected to unsupervised cluster analysis using Cluster 3.0, and the results of the hierarchical clustering were visualized using Treeview. ...
Hypermethylation of CpG island is an epigenetic event prevalent in human gliomas. Here we employed a high-throughput microarray approach for a global search of DNA methylation to identify novel epigenetic loci in specific glioma subtypes. Hierarchical clustering analysis separated 20 glioma samples according to their WHO histopathological subtypes – pilocytic astrocytomas (PAs, grade I), oligoastrocytomas (OAs, grade II) and glioblastomas (GBMs, grade IV), based on their unique methylation patterns. The overall methylation frequency of the low-grade PAs was significantly less than that of the more aggressive OAs and GBMs (0.45% versus 2.0% and 1.4%; PAs versus OAs, p<0.01; PAs versus GBMs, p<0.01). The lower level of DNA methylation observed in PAs may be in part due to the increased methylation of multiple CpG islands which occur in more advanced tumors. However, the young age of onset of PAs may also contribute to this observed difference. Although there were many hypermethylated loci exclusive to the OA and GBM subtypes, the methylation frequencies between these groups were not significantly different. Analysis by methylation-specific PCR on an expanded set of samples and on more glioma subtypes further confirmed an epigenetic marker, SMARCA5, the hypermethylation of which was preferentially observed in grade IV, but not in grades I or II gliomas (p<0.0001). The intermediate grade III gliomas showed low levels of SMARCA5 hypermethylation. Epigenetic loci uncovered in the present study recapitulate the histopathological differences of these gliomas, indicating that these molecular changes may be responsible for the development of the different glioma subtypes. Ongoing work in our laboratory has shown that some of these loci are indeed hypermethylated in the early stages of astrocytic tumors.
... Furthermore, ectopic expression of glypican-3 inhibited the growth of eight out of ten breast cancer cell lines, suggesting that glypican-3 can act as an inhibitor of breast cancer growth [329]. The hypermethylation of the glypican-3 promoter in breast cancer was confirmed by a more extensive study that showed that this promoter was hypermethylated in 38 of 45 breast tumors [331]. Notably, this study reported that high levels of glypican-3 promoter methylation are more predominant in hormone receptor-negative patients. ...
Proteoglycans control numerous normal and pathological processes, among which are morphogenesis, tissue repair, inflammation, vascularization and cancer metastasis. During tumor development and growth, proteoglycan expression is markedly modified in the tumor microenvironment. Altered expression of proteoglycans on tumor and stromal cell membranes affects cancer cell signaling, growth and survival, cell adhesion, migration and angiogenesis. Despite the high complexity and heterogeneity of breast cancer, the rapid evolution in our knowledge that proteoglycans are among the key players in the breast tumor microenvironment suggests their potential as pharmacological targets in this type of cancer. It has been recently suggested that pharmacological treatment may target proteoglycan metabolism, their utilization as targets for immunotherapy or their direct use as therapeutic agents. The diversity inherent in the proteoglycans that will be presented herein provides the potential for multiple layers of regulation of breast tumor behavior. This review summarizes recent developments concerning the biology of selected proteoglycans in breast cancer, and presents potential targeted therapeutic approaches based on their novel key roles in breast cancer.
Copyright © 2015. Published by Elsevier B.V.
... Location analysis of 5-mC was initially examined by endonuclease digestion combined with gel electrophoresis detection in the early day [85,86]. Subsequently, microarray hybridization techniques from the gene expression fields were applied to the profiling of DNA methylation patterns [87][88][89]. The advance in sequencing technologies accelerates and revolutionizes the genome-wide distribution studies of DNA methylation analysis [90,91]. ...
Epigenetics has undergone an explosion in the past decade. DNA methylation, consisting of the addition of a methyl group at the fifth position of cytosine (5-methylcytosine, 5-mC) in a CpG dinucleotide, is a well-recognized epigenetic mark with important functions in cellular development and pathogenesis. Numerous studies have focused on the characterization of DNA methylation marks associated with disease development as they may serve as useful biomarkers for diagnosis, prognosis, and prediction of response to therapy. Recently, novel cytosine modifications with potential regulatory roles such as 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC) have been discovered. Study of the functions of 5-mC and its oxidation derivatives promotes the understanding of the mechanism underlying association of epigenetic modifications with disease biology. In this respect, much has been accomplished in the development of methods for the discovery, detection, and location analysis of 5-mC and its oxidation derivatives. In this review, we focus on the recent advances for the global detection and location study of 5-mC and its oxidation derivatives 5-hmC, 5-foC, and 5-caC.
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... Differential methylation hybridisation using CpG island microarrays has actually been successfully used by many investigators to reveal differentially methylated genes in many tumour sites Yan et al, 2001;Wei et al, 2002;van Doorn et al, 2005;Wang et al, 2005;Lu et al, 2009;Lu et al, 2010). However, it is interesting to note that virtually all the published studies have utilised custom CpG island arrays. ...
... Arrays constructed from a CGI library (23) have been applied successfully to study DNA methylation status and to identify genomic fragments immunoprecipitated with antibodies against several target proteins (24,76,(99)(100)(101)(102). The rationale for using a CGI array to analyze ChIP DNA is based on the association between CGIs and gene TSSs (87,103) as well as the hypothesis that CpG conservation in these regions results from protein-chromatin interactions which prevent methylation and subsequent CpG degeneration (89). ...
... A total 2,304 mouse CpG islands library (mCGI) clones were spotted on UltraGAPS Coated Slides (Corning, MA, USA) by the BioDot AD1500 (BIODOT, CA, USA). The amplicons for methylation analysis were prepared as previously described [47,48]. ...
Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.
... Genome-wide methylation studies in lymphomas have progressed rapidly in light of the development of genome-wide technologies. Some of the earlier microarray platforms allowed for the investigation of DNA methylation present within select CpG islands [52,53]. The coverage of these arrays was limited but facilitated the discovery of putative tumor suppressor genes that are methylated in lymphomas and leukemias. ...
Epigenetic modifications play an important role in lymphoid malignancies. This has been evidenced by the large body of work published using microarray technologies to generate methylation profiles for numerous types and subtypes of lymphoma and leukemia. These studies have shown the importance of defining the epigenome so that we can better understand the biology of lymphoma. Recent advances in DNA sequencing technology have transformed the landscape of epigenomic analysis as we now have the ability to characterize the genome-wide distribution of chromatin modifications and DNA methylation using next-generation sequencing. To take full advantage of the throughput of next-generation sequencing, there are many methodologies that have been developed and many more that are currently being developed. Choosing the appropriate methodology is fundamental to the outcome of next-generation sequencing studies. In this review, published technologies and methodologies applicable to studying the methylome are presented. In addition, progress towards defining the methylome in lymphoma is discussed and prospective directions that have been made possible as a result of next-generation sequencing technology. Finally, methodologies are introduced that have not yet been published but that are being explored in the pursuit of defining the lymphoma methylome.
Genomic data from microarray studies that were available from GEO Database NCBI, were collected. The data included stromal cells, pre osteoblasts, osteoblasts, osteocytes and macrophage precursor cells from bone marrow cells that were cultured in space or ground based facilities under real or simulated microgravity, 1g or 2g gravity. Bioinformatics analysis revealed significant relation between stromal cells cultured for 192h and osteocytes cultured for 48h in 0g. Furthermore, osteocytes showed common expression pattern among 0g, 1g and 2g regardless of concomitant factors. All cell types presented an expression pattern amongst 0g, 1g and 2g gravity that moved on a certain level, thus affording predictive value. Genes that reached statistical significance were analyzed using Gene ontology and KEGG pathways, for their implication in metabolic pathways. The later, participated in MAPK signaling, osteoclast differentiation, neuronal intercommunication, purine and tryptophan metabolism, toxoplasmosis, cancer pathways, immune reactions and cytokine production. Tnfrs1a (Tumor necrosis factor receptor member 1), Il1r1 (Interleukin 1 receptor type 1), MAPK9 (mitogen-activated protein kinase 9), MAP2K7 (mitogen-activated protein kinase 7) were expressed in more than two of the aforementioned pathways. These genes are implicated in inflammatory responses, immune system activation (IgE mediated), osteoclast differentiation and MAPK signaling,, causing activation of NF kB transcription factor complex. Therefore, osteoclast activation is mediated directly or via inflammatory routes, resulting in osteolysis.
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Cancer stem cells (CSCs) have been suggested as central precursor of tumor initiation, progression, recurrence, and therapeutic impedance. Thus, identifying stem cells like moities within cancers and understanding their properties is important for the development of efficacious anticancer therapies and diagnostics. Lysed cancer stem cells can be analyze at genome, transcriptome, and proteome level from a single biological sample can provide better understanding. In this review, we have summarized major techniques that can be useful for cancer stem cell expression profiling at genome level, transcriptome and proteome level. Cancer biomarker based genome analysis can provide cancer diagnosis, prognostic, pharmacodynamics. Microarray based transcriptome profiling can provide better understanding at RNA level and also post transcription modifications, that are responsible for cancer. Proteomics techniques include gel-free and gel-based methods. Gel free method include stable isotope labeling by amino acids in cell culture (SILAC), Isobaric tags for relative and absolute quantitation (iTRAQ) and isotope coded affinity tagging (ICAT) is upgraded and better technique for Chemical proteomic, a combination of isotope coded affinity tagging (ICAT) and mass spectrometry, have proposed as powerful tools for identifying drug targets and explaining complex latent mechanisms of drug action against CSCs. Extracted protein can be analyzed by liquid chromatography (LC) coupled to MS/MS. This detection method permits for high resolution separation, thereby precisely revealing differential protein expression profiles. Gel-based methods employ, Two-dimensional difference in gel electrophoresis (2D-DIGE) is a method in which protein samples labeled by different fluorescent dyes are mixed with identical concentrations. A comparison generated 2D-DIGE fluorescence images allows for quantification of each spot. Hence, 2D-DIGE can reduce gel-to-gel variation by allowing simultaneous separation and comparison of several samples on one gel. Molecularly targeted drug delivery by Immunoliposomes, in which molecular targeting of cancer cells via liposomes conjugated with antibody or other ligand.
Epigenetics literally means upon genetics and comprises various mechanisms that regulate both gene expression and genome stability without modifying the DNA sequence itself. The most widely studied epigenetic changes are DNA methylation of cytosines within CpG dinucleotides and a growing number of chemical modifications at different amino acid residues of histone tails including acetylation, methylation, phosphorylation, and ubiquitination. Additionally, other epigenetic factors like nuclear positioning, noncoding RNAs, and microRNAs are also associated with gene regulation and chromatin structure.
Cancer results from genetic and epigenetic aberrations, many of which alter the levels of expressed genes and proteins. In the past, cancer researchers have studied genes mainly one at a time. Increasingly, researchers are using genomic technologies like DNA microarrays and next-generation DNA sequencing to investigate thousands of genes simultaneously, defining genes and pathways relevant to cancer mechanisms, diagnosis, prognostication, and treatment. This chapter details genomics technologies, their use and applications in cancer research, and their potential utility in oncologic practice.
DNA methylation is an epigenetic modification of DNA, where a methyl group is added at the fifth carbon of the cytosine base to form 5 methyl cytosine (5mC) without altering the DNA sequences. It plays important roles in regulating many cellular processes by modulating key genes expression. Alteration in DNA methylation patterns becomes particularly important in the aetiology of different diseases including cancers. Abnormal methylation pattern could contribute to the pathogenesis of cancer either by silencing key tumor suppressor genes or by activating oncogenes. Thus, DNA methylation biosensing can help in the better understanding of cancer prognosis and diagnosis and aid the development of therapies. Over the last few decades, a plethora of optical detection techniques have been developed for analyzing DNA methylation using fluorescence, Raman spectroscopy, surface plasmon resonance (SPR), electrochemiluminescence and colorimetric readouts. This paper aims to comprehensively review the optical strategies for DNA methylation detection. We also present an overview of the remaining challenges of optical strategies that still need to be focused along with the lesson learnt while working with these techniques.
DNA methylation is an epigenetic modification of DNA, where a methyl group is added at the fifth carbon of the cytosine base to form 5 methyl cytosine (5mC) without altering the DNA sequences. It plays important roles in regulating many cellular processes by modulating key genes expression. Alteration in DNA methylation patterns becomes particularly important in the aetiology of different diseases including cancers. Abnormal methylation pattern could contribute to the pathogenesis of cancer either by silencing key tumor suppressor genes or by activating oncogenes. Thus, DNA methylation biosensing can help in the better understanding of cancer prognosis and diagnosis and aid the development of therapies. Over the last few decades, a plethora of optical detection techniques have been developed for analyzing DNA methylation using fluorescence, Raman spectroscopy, surface plasmon resonance (SPR), electrochemiluminescence and colorimetric readouts. This paper aims to comprehensively review the optical methylation strategies for DNA methylation detection. We also present an overview of the remaining challenges of optical strategies that still need to be focused along with the lesson learnt while working with these techniques.
We present novel optimization-based classification models that are of general purpose and suitable for establishing reliable predictive rules for a broad variety of biological and medical applications. Our predictive framework simultaneously incorporates (i) the ability to classify any number of distinct groups; (ii) the ability to incorporate heterogeneous and temporal types of attributes as input; (iii) a high-dimensional data transformation that minimizes noise and errors; (iv) the ability to incorporate constraints to limit the rate of misclassification, and a reserved-judgment region that provides a safeguard against overtraining (which tends to lead to high misclassification rates from the resulting predictive rule); and (v) successive multistage classification capability to handle data points placed in the reserved-judgment region. To illustrate the power and flexibility of the classification model and solution engine and its multigroup prediction capability, applications of the predictive model to a broad class of biological and medical problems are described. Applications include vaccine immunity prediction; early detection of mild cognitive impairment (MCI) and Alzheimer's disease (AD); predicting aberrant CpG island methylation in human; predicting ultrasonic cell disruption for drug delivery; uncovering tumor shape and volume in the treatment of sarcoma; multistage discriminant analysis of biomarkers for detecting early atherosclerosis; and fingerprinting of native and angiogenic microvascular networks for early diagnosis of diabetes, aging, macular degeneracy, and tumor metastasis. Furthermore, we also demonstrate the classification performance on instances from the UCI Repository of machine learning databases. In all these applications, the classification rules yield blind prediction accuracy ranging from 80% to 100%. This provides motivation for pursuing its use as a medical diagnostic, monitoring, and decision-making tool.
We review the profound effects that components of diets commonly consumed in western societies and linked through population studies to risk for colon cancer have on the development of intestinal cancer in humans and in mouse models. Focus is particularly on levels of vitamin D, interactive with calcium and fat, in establishing probability of tumor development even in mouse genetic models in which there is high penetrance of the disease. These dietary factors have also been used to develop a mouse model of dietary-induced sporadic colon cancer which exhibits similar lag, incidence, and frequency of tumor development, and relative incidence of carcinomas and adenomas, as seen for >90% of colon tumors that arise in the general population later in life. Potential mechanisms influenced by diet that alter probability of tumor development are outlined, including altered patterns of intermediary metabolism, differentiation, and inflammation in the intestinal mucosa, all apparent in the histopathologically normal intestinal mucosa well before neoplastic changes become detectable. This includes pathways by which macrophages signal to intestinal epithelial cells, revealing a new paradigm for how vitamin D may influence tumor development.
Genomic, chromosomal, and gene-specific changes in the DNA sequence underpin both phenotypic variations in populations as well as disease associations, and the application of genomic technologies for the identification of constitutional (inherited) or somatic (acquired) alterations in DNA sequence forms a cornerstone of clinical and molecular genetics. In addition to the disruption of primary DNA sequence, the modulation of DNA function by epigenetic phenomena, in particular by DNA methylation, has long been known to play a role in the regulation of gene expression and consequent pathogenesis. However, these epigenetic factors have been identified only in a handful of pediatric conditions, including imprinting disorders. Technological advances in the past decade that have revolutionized clinical genomics are now rapidly being applied to the emerging discipline of clinical epigenomics. Here, we present an overview of epigenetic mechanisms with a focus on DNA modifications, including the molecular mechanisms of DNA methylation and subtypes of DNA modifications, and we describe the classic and emerging genomic technologies that are being applied to this study. This review focuses primarily on constitutional epigenomic conditions associated with a spectrum of developmental and intellectual disabilities. Epigenomic disorders are discussed in the context of global genomic disorders, imprinting disorders, and single gene disorders. We include a section focused on integration of genetic and epigenetic mechanisms together with their effect on clinical phenotypes. Finally, we summarize emerging epigenomic technologies and their impact on diagnostic aspects of constitutional genetic and epigenetic disorders.
The sections in this article are
Introduction
Types of Microarrays
DNA Microarrays
Protein Microarrays
Tissue Microarrays
Cell‐Based Microarrays (Transfection Microarrays)
Principle Steps of an Experimental Microarray Process
Basics
Surface Biofunctionalization
Preparation of Biomolecules (Sample Preparation)
DNA
RNA
Proteins
Target Amplification and Labeling
PCR – Polymerase Chain Reaction
In Vitro Transcription – c DNA / IVT
Bio‐Barcode Amplification of Nucleic Acids and Proteins
Radioactive and Fluorophore Labeling
Enzymes
Nanoparticles
Readout/Detection
Radioactivity
Fluorescence/Absorbance
Surface Plasmon Resonance
Examples of Biochip Applications in Biology and Medicine
Genomic Research
SNP Detection
Gene Expression Profile Analysis
Pharmacogenomics and Toxicogenomics
Pathogen Detection and Clinical Diagnosis
Pathogen Detection
Clinical Diagnosis
Trends in Biochip Research
Metal Nanoparticles
Quantum Dots
Enzymes
Conclusion
This chapter discusses the implications that epigenetic mechanisms have in the progression of cancer, the importance of epigenetics for angiogenesis and metastasis, and the assays useful for evaluating angiogenesis and metastasis in experimental settings. Epigenetic control of gene expression and its importance in cancer and disease are described in the chapter. Angiogenesis is the formation of blood vessels, from the preexisting vascular architecture. Angiogenesis allows growing tumors to recruit their own blood supply. It is also important in hematologic malignancies, where new blood vessel growth is present in the bone marrow and the lymph nodes of patients, with acute lymphoblastic leukemia and B-cell nonHodgkins lymphoma. The metastatic process is divided into several steps, each of which is thought to be rate-limiting, such as primary malignant neoplasm or neovascularisation. Assays, allowing the analysis of functional ability to metastasize, are also developed. Gene products involved in angiogenesis, cell migration, attachment, and survival are all potential targets for epigenetic regulation, by CpG methylation and histone modifications. Therapeutic agents are designed to target the epigenetic effects, either by altering methylation patterns on DNA or modifying histones.
The suffix -omics is a neologism that has been added to the terms of many areas, primarily in life sciences and technology, to denote studies undertaken on a large or genome-wide scale. The first terms were genomics, transcriptomics, and proteomics, which have been used to define large-scale gene expression profiles. The number of -omics have been growing ever since, as an analogy from the first genomic studies. Epigenetics refers to the study of changes in gene expression caused by mechanisms other than changes in DNA sequence. Epigenetic mechanisms include DNA methylation, histone modifications, and, more recently, a variety of noncoding RNAs. Hence, the word epigenomics designates the complete set of epigenetic modifications that occur across the entire genome of a cell at a specific developmental time. Here we review different methodologies to analyze the epigenetic modifications genome-wide (whole methylome and histone-maps), detailing advantages and limitations for each approach.
DNA methylation is an important mode of epigenetic modification and has great significance in biochemistry, medicine and genomics. The development of techniques for DNA-methylation analysis is fundamental and pivotal for epigenome research. In around 30 years of diligent study, direct and indirect approaches were developed. Direct approaches mainly focus on global methylation analysis, while indirect approaches rely on specific recognition of DNA-methylation loci. In this review, we introduce typical methods, especially those based on developing the method of methylation-loci recognition from the most used, bisulfite conversion, biological recognition, and the newly proposed chemical cleavage. Furthermore, we assess the perspectives on the research methodology of DNA-methylation analysis. To date, there have been few summaries of the research methodology of DNA-methylation analysis. The review is envisioned to be helpful for the researchers to design novel methods of DNA-methylation analysis.
This paper reports the first electrochemical assessment of the cytosine methylation status in genomic DNA by using a combined bisulfite restriction analysis on a nanocarbon film electrode. The film electrode consists of sp2 and sp3 hybrid nanocarbon, which is fabricated with unbalanced magnetron sputtering equipment and is suitable for cadmium ion determination. The electrochemical assessment was performed by measuring the oxidation current with an anodic stripping voltammogram derived from the labeled cadmium-containing nanoparticles, following digestion by a restriction enzyme (HpyCH4IV). We succeeded in estimating the methylation ratio at a specific CpG site from genomic DNA in a diluted PCR product solution from 0.5 to 10 nM.
Deregulation of the epigenome is an important mechanism involved in the development and progression of human diseases such as cancer. As opposed to the irreversible nature of genetic events, which introduce changes in the primary DNA sequence, epigenetic modifications are reversible. The conventional analysis of neo-plasias, however, has preferentially focused on elucidating the genetic contribution to tumorigenesis, which has resulted in a biased and incomplete understanding of the mechanisms involved in tumor formation. Epigenetic alterations, such as aberrant DNA methylation and altered histone modifications, are not only sufficient to induce tumors, but can also modify tumor incidence and even determine the type of neoplasia that will arise in genetic models of cancer. There is clear evidence that the epigenetic landscape in humans undergoes modifications as the result of normal aging. Thus, it has been proposed that the higher incidence of certain disease in older individuals might be, in part, a consequence of an inherent change in the regulation of the epigenome. These observations raise important questions about the degree to which genetic and epigenetic mechanisms cooperate in human tumorigenesis, the identity of the specific cooperating genes, and how these genes interact functionally to determine the diverse biological paths to tumor initiation and progression. The answers to these questions will partially rely on sequencing relevant regions of the 3 billion nucleotide genome, and determining the methylation status of the 30 million CpG dinucleotide methylome at single nucleotide resolution in different types of neoplasias. Here, we also review the emergence and advancement of technologies to map ever larger proportions of the cancer methylome, and the unique discovery potential of integrating these technologies with cancer genomic data. We discuss the knowledge gained from these large-scale analyses in the context of gene discovery, therapeutic application, and building a more widely applicable mechanism-based model of human tumorigenesis.
It is widely believed that ductal breast cancer dissemination involves a succession of clinical and pathological stages starting with carcinoma in situ, progressing into invasive lesion and culminating in metastatic disease. Such changes have frequently been attributed to the sequential acquisition of various alterations in a single cell followed by clonal selection and expansion, thus leading to intratumor diversity. According to this multi-step view, extensive genotype and phenotype (marker expression, grade) shift may occur in the same tumor during progression; this may lead to the coexistence of molecularly and/or pathologically different areas within the same lesion. An increasing amount of data of various natures now appear to challenge this concept: only a few distinct ‘portraits’, in relation to estrogen receptor (ER) status and grade, may be found among tumors. Moreover, although undergoing increasing genetic alteration, most individual lesions largely maintain their phenotype when they evolve from in situ to the metastatic state. While many of the data presented here are related to ductal tumors, lobular cancer is also discussed.
DNA methylation is the most studied epigenetic event in cancer, with focus being placed on studying the entire DNA methylation landscape in specific cancers. Due to the recent advances of next-generation sequencing technology, several effective methods have been developed for high-throughput analysis of DNA methylation, enabling DNA methylation markers to be innovative diagnostic and therapeutic strategies in cancer. In this review, we discuss various current and emerging technologies in DNA methylation analysis that integrate next-generation sequencing with the basic principles of methylation detections including methylation sensitive restriction enzyme digestion, affinity purification with antibody or binding proteins, and bisulfite treatment. Variations to these described methods have also allowed for detection of 5-hydroxymethylcytosine marks on a genome-wide scale. We also describe several of the bioinformatic tools used to properly analyze methylome-sequencing data. Finally, recently developed artificial transcription-factor (ATF) targeting tools may provide flexible manipulation of DNA methylation events in specific gene regions, revealing the functional consequences of particular DNA methylation events.
Optimization has become an essential tool in addressing the limitation of resources and need for better decision-making in the medical field. Both continuous and discrete mathematical techniques are playing an increasingly important role in understanding several fundamental problems in medicine.
This volume presents a wide range of medical applications that can utilize mathematical computing. Examples include using an algorithm for considering the seed reconstruction problem in brachytherapy and using optimization-classification models to assist in the early prediction, diagnosis and detection of diseases. Discrete optimization techniques and measures derived from the theory of nonlinear dynamics, with analysis of multi-electrode electroencephalographic (EEG) data, assist in predicting impending epileptic seizures. Mathematics in medicine can also be found in recent cancer research. Sophisticated mathematical models and optimization algorithms have been used to generate treatment plans for radionuclide implant and external beam radiation therapy. Optimization techniques have also been used to automate the planning process in Gamma Knife treatment, as well as to address a variety of medical image registration problems.
This work grew out of a workshop on optimization which was held during the 2005 CIM Thematic Term on Optimization in Coimbra, Portugal. It provides an overview of the state-of-the-art in optimization in medicine and will serve as an excellent reference for researchers in the medical computing community and for those working in applied mathematics and optimization.
Microarray data feature selection is crucial for the development of a viable cancer diagnostic system based on microarray data. This paper assesses the effectiveness of the Hybrid Genetic Algorithm Simulated Annealing (HGASA) algorithm in selecting features for various classification architectures. HGASA combines the parallel search capability of Genetic Algorithm (GA) with the flexibility of Simulated Annealing (SA). The algorithm is guided by Separability Index, which quantifies the extent of class separability demonstrated by a combination of features. Four classifiers are used in the assessment: Artificial Neural Network (ANN), Support Vector Machine (SVM), Naïve Bayesian Classifier (NBC) and K-Nearest Neighbour (KNN) classifier. Results from HGSA is compared to those from standard GA as well as to those from Population based incremental Learning (PBIL) algorithm. Two data sets are used facilitate this analysis: a prostate cancer data set and a lymphoma data set. For the prostate cancer data set, features selected by the HGASA attained the highest classification accuracy on the SVM classifier with an accuracy of 88%. For the Lymphoma data set, the highest classification accuracy was attained using the ANN classifier, which attained an accuracy of 95%. The performance of the HGASA is ascribed to its ability to search the feature space more thoroughly by employing a deeper exploration of the feature space, when compared to GA and PBIL.
: Epigenetic regulation of gene expression has been observed in a variety of tumor types. We have used microarray technology to evaluate the predisposition of drug response by aberrant methylation in ovarian cancer. Results indicate that loss of gene activity due to hypermethylation potentially confers a predisposition in certain cancer types and is an early event in disease progression. Methylation profiles of ovarian cancer might be useful for early cancer detection and prediction of chemotherapy outcome in a clinical context.
Post-translational modifications of DNA- changes in the chemical structure of individual bases that occur without changes in the DNA sequence- are known to alter gene expression. They are believed to result in frequently deleterious phenotypic changes, such as cancer. Methylation of adenine, methylation and hydroxymethylation of cytosine, and guanine oxidation are the primary DNA base modifications identified to date. Here we show it is possible to use surface enhanced Raman spectroscopy (SERS) to detect these primary DNA base modifications. SERS detection of modified DNA bases is label-free and requires minimal additional sample preparation, reducing the possibility of additional chemical modifications induced prior to measurement. This approach shows the feasibility of DNA base modification assessment as a potentially routine analysis that may be further developed for clinical diagnostics.
We are in an era where the potential exists for deriving comprehensive profiles of DNA alterations characterizing each form of human cancer. Such profiles would provide invaluable insight into mechanisms underly- ing the evolution of each tumor type and will provide molecular markers, which could radically improve cancer detection. To date, no one type of DNA change has been defined which accomplishes this purpose. Herein, by using a candidate gene approach, we show that one category of DNA alteration, aberrant methylation of gene promoter regions, can enor- mously contribute to the above goals. We have now analyzed a series of promoter hypermethylation changes in 12 genes (p16INK4a, p15INK4b, p14ARF, p73, APC,5 BRCA1, hMLH1, GSTP1, MGMT, CDH1, TIMP3, and DAPK), each rigorously characterized for association with abnormal gene silencing in cancer, in DNA from over 600 primary tumor samples rep- resenting 15 major tumor types. The genes play known important roles in processes encompassing tumor suppression, cell cycle regulation, apopto- sis, DNA repair, and metastastic potential. A unique profile of promoter hypermethylation exists for each human cancer in which some gene changes are shared and others are cancer-type specific. The hypermethy- lation of the genes occurs independently to the extent that a panel of three to four markers defines an abnormality in 70 -90% of each cancer type. Our results provide an unusual view of the pervasiveness of DNA alter- ations, in this case an epigenetic change, in human cancer and a powerful set of markers to outline the disruption of critical pathways in tumori- genesis and for derivation of sensitive molecular detection strategies for virtually every human tumor type.
Methylation of CpG islands is an established transcriptional repressive mechanism and is a feature of silencing in X chromosome
inactivation. Housekeeping genes that are subject to X inactivation exhibit differential methylation of their CpG islands
such that the inactive alleles are hypermethylated. In this report, we examine two contrasting X-linked genes with CpG islands
for regulation by DNA methylation: SYBL1, a housekeeping gene in the Xq pseudoautosomal region, and GPC3, a tissue-specific gene in Xq26 that is implicated in the etiology of the Simpson–Golabi–Behmel overgrowth syndrome. We observed
that in vitro methylation of either the SYBL1 or the GPC3 promoter resulted in repression of reporter constructs. In normal contexts, we found that both the Y and inactive X alleles
of SYBL1 are repressed and hypermethylated, whereas the active X allele is expressed and unmethylated. Furthermore, the Y and inactive
X alleles of SYBL1 were derepressed by treatment with the demethylating agent azadeoxycytidine. GPC3 is also subject to X inactivation, and the active X allele is unmethylated in nonexpressing leukocytes as well as in an expressing
cell line, suggesting that methylation is not involved in the tissue-specific repression of this allele. The inactive X allele,
however, is hypermethylated in leukocytes, presumably reflecting early X inactivation events that become important for gene
dosage in expressing lineages. These and other data suggest that all CpG islands on Xq, including the pseudoautosomal region,
are subject to X inactivation-induced methylation. Additionally, methylation of SYBL1 on Yq may derive from a process related to X inactivation that targets large chromatin domains for transcriptional repression.
In contrast to earlier views that there was much compartmentalization of the types of sequences subject to cancer-linked changes in DNA epigenetics, it is now clear that both cancer-associated DNA hypomethylation and hypermethylation are found throughout the genome. The hypermethylation includes promoters of tumor suppressor genes whose expression becomes repressed, thereby facilitating cancer formation. How hypomethylation contributes to carcinogenesis has been less clear. Recent insights into tissue-specific intra- and intergenic methylation and into cancer methylomes suggest that some of the DNA hypomethylation associated with cancers is likely to aid in tumor formation and progression by many different pathways, including effects on transcription in cis. Cancer-associated loss of DNA methylation from intergenic enhancers, promoter regions, silencers, and chromatin boundary elements may alter transcription rates. In -addition, cancer-associated intragenic DNA hypomethylation might modulate -alternative promoter usage, -production of intragenic noncoding RNA transcripts, cotranscriptional splicing, and transcription initiation or elongation. Initial studies of hemimethylation of DNA in cancer and many new studies of DNA demethylation in normal tissues suggest that active demethylation with spreading of hypomethylation can explain much of the cancer-associated DNA hypomethylation. The new discoveries that genomic 5-hydroxymethylcytosine is an intermediate in DNA demethylation, a base with its own functionality, and a modified base that, like 5-methylcytosine, exhibits cancer-associated losses, suggest that both decreased hydroxymethylation and decreased methylation of DNA play important roles in carcinogenesis.
Gene function in cancer can be disrupted either through genetic alterations, which directly mutate or delete genes, or epigenetic alterations, which alter the heritable state of gene expression. The latter events are mediated by formation of transcriptionally repressive chromatin states around gene transcription start sites and an associated gain of methylation in normally unmethylated CpG islands in these regions. The genes affected include over half of the tumor suppressor genes that cause familial cancers when mutated in the germline; the selective advantage for genetic and epigenetic dysfunction in these genes is very similar. The aberrant methyla-tion can begin very early in tumor progression and mediate most of the important pathway abnormalities in cancer including loss of cell cycle control, altered function of transcription factors, altered receptor function, disruption of normal cell–cell and cell–substratum interaction, inactivation of signal transduction pathways, loss of apoptotic signals and genetic instability. The active role of the aberrant methylation in transcriptional silencing of genes is becoming increasingly understood and involves a synergy between the methylation and histone deacetylase (HDAC) activity. This synergy can be mediated directly by HDAC interaction with DNA methylating enzymes and by recruitment through complexes involving methyl-cytosine binding proteins. In the translational arena, the promoter hypermethylation changes hold great promise as DNA tumor markers and their potentially reversible state creates a target for cancer therapeutic strategies involving gene reactivation.
CpG islands are short stretches of DNA containing a high density of non-methylated CpG dinucleotides, predominantly associated with coding regions. We have constructed an affinity matrix that contains the methyl-CpG binding domain from the rat chromosomal protein MeCP2, attached to a solid support. A column containing the matrix fractionates DNA according to its degree of CpG methylation, strongly retaining those sequences that are highly methylated. Using this column, we have developed a procedure for bulk isolation of CpG islands from human genomic DNA. As CpG islands overlap with approximately 60% of human genes, the resulting CpG island library can be used to isolate full-length cDNAs and to place genes on genomic maps.
Methylation of CpG islands is an established transcriptional repressive mechanism and is a feature of silencing in X chromosome inactivation. Housekeeping genes that are subject to X inactivation exhibit differential methylation of their CpG islands such that the inactive alleles are hypermethylated. In this report, we examine two contrasting X-linked genes with CpG islands for regulation by DNA methylation: SYBL1, a housekeeping gene in the Xq pseudoautosomal region, and GPC3, a tissue-specific gene in Xq26 that is implicated in the etiology of the Simpson-Golabi-Behmel overgrowth syndrome. We observed that in vitro methylation of either the SYBL1 or the GPC3 promoter resulted in repression of reporter constructs. In normal contexts, we found that both the Y and inactive X alleles of SYBL1 are repressed and hypermethylated, whereas the active X allele is expressed and unmethylated. Furthermore, the Y and inactive X alleles of SYBL1 were derepressed by treatment with the demethylating agent azadeoxycytidine. GPC3 is also subject to X inactivation, and the active X allele is unmethylated in nonexpressing leukocytes as well as in an expressing cell line, suggesting that methylation is not involved in the tissue-specific repression of this allele. The inactive X allele, however, is hypermethylated in leukocytes, presumably reflecting early X inactivation events that become important for gene dosage in expressing lineages. These and other data suggest that all CpG islands on Xq, including the pseudoautosomal region, are subject to X inactivation-induced methylation. Additionally, methylation of SYBL1 on Yq may derive from a process related to X inactivation that targets large chromatin domains for transcriptional repression.
The discovery of numerous hypermethylated promoters of tumour-suppressor genes, along with a better understanding of gene-silencing mechanisms, has moved DNA methylation from obscurity to recognition as an alternative mechanism of tumour-suppressor inactivation in cancer. Epigenetic events can also facilitate genetic damage, as illustrated by the increased mutagenicity of 5-methylcytosine and the silencing of the MLH1 mismatch repair gene by DNA methylation in colorectal tumours. We review here current mechanistic understanding of the role of DNA methylation in malignant transformation, and suggest Knudson's two-hit hypothesis should now be expanded to include epigenetic mechanisms of gene inactivation.
Although cancer classification has improved over the past 30 years, there has been no general approach for identifying new cancer classes (class discovery) or for assigning tumors to known classes (class prediction). Here, a generic approach to cancer classification based on gene expression monitoring by DNA microarrays is described and applied to human acute leukemias as a test case. A class discovery procedure automatically discovered the distinction between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) without previous knowledge of these classes. An automatically derived class predictor was able to determine the class of new leukemia cases. The results demonstrate the feasibility of cancer classification based solely on gene expression monitoring and suggest a general strategy for discovering and predicting cancer classes for other types of cancer, independent of previous biological knowledge.
Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.
Simpson-Golabi-Behmel syndrome (SGBS) is an X-linked syndrome characterized by pre- and postnatal overgrowth (gigantism), which clinically resembles the autosomal Beckwith-Wiedemann syndrome (BWS). Deletions and translocations involving the glypican-3 gene ( GPC3 ) have been shown to be associated with SGBS. Occasionally, these deletions also include the glypican-4 gene ( GPC4 ). Glypicans are heparan sulfate proteoglycans which have a role in the control of cell growth and cell division. We have examined the mutational status of the GPC3 and GPC4 genes in one patient with Perlman syndrome, three patients with overgrowth without syndrome diagnosis, ten unrelated SGBS-patients and 11 BWS patients. We identified one SGBS patient with a deletion of a GPC3 exon. Six SGBS patients showed point mutations in GPC3. One frameshift, three nonsense, and one splice mutation predict a loss-of-function of the glypican-3 protein. One missense mutation, W296R, changes an amino acid that is conserved in all glypicans identified so far. A GPC3 protein that reproduces this mutation is poorly processed and fails to increase the cell surface expression of heparan sulfate, suggesting that this missense mutation is also a loss-of-function mutation. In three SGBS patients and in all non-SGBS patients, no mutations could be identified. We found three single nucleotide polymorphisms in the GPC4 gene but no evidence for loss-of-function mutations in GPC4 associated with SGBS.
The most common human cancers are malignant neoplasms of the skin. Incidence of cutaneous melanoma is rising especially steeply, with minimal progress in non-surgical treatment of advanced disease. Despite significant effort to identify independent predictors of melanoma outcome, no accepted histopathological, molecular or immunohistochemical marker defines subsets of this neoplasm. Accordingly, though melanoma is thought to present with different 'taxonomic' forms, these are considered part of a continuous spectrum rather than discrete entities. Here we report the discovery of a subset of melanomas identified by mathematical analysis of gene expression in a series of samples. Remarkably, many genes underlying the classification of this subset are differentially regulated in invasive melanomas that form primitive tubular networks in vitro, a feature of some highly aggressive metastatic melanomas. Global transcript analysis can identify unrecognized subtypes of cutaneous melanoma and predict experimentally verifiable phenotypic characteristics that may be of importance to disease progression.
Human breast tumours are diverse in their natural history and in their responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome. Here we have characterized variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals, using complementary DNA microarrays representing 8,102 human genes. These patterns provided a distinctive molecular portrait of each tumour. Twenty of the tumours were sampled twice, before and after a 16-week course of doxorubicin chemotherapy, and two tumours were paired with a lymph node metastasis from the same patient. Gene expression patterns in two tumour samples from the same individual were almost always more similar to each other than either was to any other sample. Sets of co-expressed genes were identified for which variation in messenger RNA levels could be related to specific features of physiological variation. The tumours could be classified into subtypes distinguished by pervasive differences in their gene expression patterns.
Gene function in cancer can be disrupted either through genetic alterations, which directly mutate or delete genes, or epigenetic alterations, which alter the heritable state of gene expression. The latter events are mediated by formation of transcriptionally repressive chromatin states around gene transcription start sites and an associated gain of methylation in normally unmethylated CpG islands in these regions. The genes affected include over half of the tumor suppressor genes that cause familial cancers when mutated in the germline; the selective advantage for genetic and epigenetic dysfunction in these genes is very similar. The aberrant methylation can begin very early in tumor progression and mediate most of the important pathway abnormalities in cancer including loss of cell cycle control, altered function of transcription factors, altered receptor function, disruption of normal cell-cell and cell-substratum interaction, inactivation of signal transduction pathways, loss of apoptotic signals and genetic instability. The active role of the aberrant methylation in transcriptional silencing of genes is becoming increasingly understood and involves a synergy between the methylation and histone deacetylase (HDAC) activity. This synergy can be mediated directly by HDAC interaction with DNA methylating enzymes and by recruitment through complexes involving methyl-cytosine binding proteins. In the translational arena, the promoter hypermethylation changes hold great promise as DNA tumor markers and their potentially reversible state creates a target for cancer therapeutic strategies involving gene reactivation.
Although cancer classification has improved over the past 30 years, there has been no general approach for identifying new
cancer classes (class discovery) or for assigning tumors to known classes (class prediction). Here, a generic approach to
cancer classification based on gene expression monitoring by DNA microarrays is described and applied to human acute leukemias
as a test case. A class discovery procedure automatically discovered the distinction between acute myeloid leukemia (AML)
and acute lymphoblastic leukemia (ALL) without previous knowledge of these classes. An automatically derived class predictor
was able to determine the class of new leukemia cases. The results demonstrate the feasibility of cancer classification based
solely on gene expression monitoring and suggest a general strategy for discovering and predicting cancer classes for other
types of cancer, independent of previous biological knowledge.
Recently, the concept that epigenetic, as well as genetic, events might be central to the evolution of human cancer is re-emerging. Cancers often exhibit an aberrant methylation of gene promoter regions that is associated with loss of gene function. This DNA change constitutes a heritable state, not mediated by altered nucleotide sequence, that appears to be tightly linked to the formation of transcriptionally repressive chromatin. This epigenetic process acts as an alternative to mutations to disrupt tumor-suppressor gene function and can predispose to genetic alterations through inactivating DNA-repair genes. Dissecting the molecular processes that mediate these methylation changes will enhance our understanding of chromatin modeling and gene regulation and might present novel possibilities for cancer therapy. Methylation changes constitute potentially sensitive molecular markers to define risk states, monitor prevention strategies, achieve early diagnosis, and track the prognosis of cancer.
A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.
CpG island hypermethylation is known to be associated with gene silencing in cancer. This epigenetic event is generally accepted as a stochastic process in tumor cells resulting from aberrant DNA methyltransferase (DNA-MTase) activities. Specific patterns of CpG island methylation could result from clonal selection of cells having growth advantages due to silencing of associated tumor suppressor genes. Alternatively, methylation patterns may be determined by other, as yet unidentified factors. To explore further the underlying mechanisms, we developed a novel array-based method, called differential methylation hybridization (DMH), which allows a genome-wide screening of hypermethylated CpG islands in tumor cells. DMH was used to determine the methylation status of >276 CpG island loci in a group of breast cancer cell lines. Between 5 and 14% of these loci were hypermethylated extensively in these cells relative to a normal control. Pattern analysis of 30 positive loci by Southern hybridization indicated that CpG islands might differ in their susceptibility to hypermethylation. Loci exhibiting pre-existing methylation in normal controls were more susceptible to de novo methylation in these cancer cells than loci without this condition. In addition, these cell lines exhibited different intrinsic abilities to methylate CpG islands not directly associated with methyltransferase activities. Our study provides evidence that, aside from random DNA-MTase action, additional cellular factors exist that govern aberrant methylation in breast cancer cells.
CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.
CpG island hypermethylation is a frequent epigenetic event in cancer. We have recently developed an array-based method, called differential methylation hybridization (DMH), allowing for a genome-wide screening of CpG island hypermethylation in breast cancer cell lines (T. H-M. Huang et al., Hum. Mol. Genet., 8: 459-470, 1999). In the present study, DMH was applied to screen 28 paired primary breast tumor and normal samples and to determine whether patterns of specific epigenetic alterations correlate with pathological parameters in the patients analyzed. Amplicons, representing a pool of methylated CpG DNA derived from these samples, were used as hybridization probes in an array panel containing 1104 CpG island tags. Close to 9% of these tags exhibited extensive hypermethylation in the majority of breast tumors relative to their normal controls, whereas others had little or no detectable changes. Pattern analysis in a subset of CpG island tags revealed that CpG island hypermethylation is associated with histological grades of breast tumors. Poorly differentiated tumors appeared to exhibit more hypermethylated CpG islands than their moderately or well-differentiated counterparts (P = 0.041). This early finding lays the groundwork for a population-based DMH study and demonstrates the need to develop a database for examining large-scale methylation data and for associating specific epigenetic signatures with clinical parameters in breast cancer.
Methylation of DNA is a biochemical modification that can influence gene expression and is involved in inactivating one of the two X chromosomes in women. Evidence that has accumulated in the past 10 years suggests that cancer cells usurp this physiologic mechanism and use it to their benefit by inactivating tumor suppressor genes and related proteins. However, the primary structure of the affected proteins remains intact; reversal of abnormalities in DNA methylation may therefore restore the tumor-suppressive function of these genes and provide a novel approach to cancer therapy. Two demethylating drugs, 5-azacytidine and 5-aza-deoxycytidine, are currently being tested in clinical trials, and several others are in preclinical development. In this article, the biological rationale for targeting aberrant methylation in cancer therapy is reviewed and completed phase I and II trials of this approach, some of which show promise for treatment of hematologic malignancies, are summarized.
The aim of this study was to examine CpG island methylation patterns in ovarian cancer and determine whether epigenetic information can be related to clinical data of patients. CpG island (CpGI) hypermethylation is commonly associated with cancer progression, but little is currently known about the role of methylation in ovarian cancer.
Differential methylation hybridization (DMH) analysis at 742 loci was performed to determine methylation signatures for 20 primary epithelial ovarian carcinomas (Stages II, III, and IV adenocarcinomas, serous papillary), 6 ovarian cancer cell lines, and normal ovarian surface epithelial cells.
Between 23 and 108 methylated CpGIs were seen in the ovarian carcinomas. Fewer (P < 0.05) methylated CpGIs were observed in the ovarian cancer cell lines; however, a number of CpGIs were commonly hypermethylated in both the cell lines and the tumor samples. A methylation signature, consisting of frequently (P < 0.05) methylated CpGIs, was determined for the samples. The observed pattern of methylation in ovarian cancers included several (11) CpGI tags that were previously reported to be hypermethylated in human breast cancer.
Epigenetic signatures in ovarian cancer were determined using DMH. This proof-of-concept study lays the foundation for genome-wide screening of methylation to examine epigenotype-phenotype relationships in ovarian cancer.