ATM Mediates pRB Function To Control DNMT1 Protein Stability and DNA Methylation

The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.     

Methylation of the <Emphasis Type="Italic">RASSF1A</Emphasis> promoter region and the allelic imbalance frequencies in chromosome 3 critical regions correlate with progression of clear cell renal carcinoma

The short arm of chromosome 3 (3p) contains several critical regions that have increased frequencies of allelic deletions and harbor a set of tumor suppressor genes. In particular, the range of functions performed by RASSF1A (LUCA region, 3p21.31) includes those potentially associated with carcinogenesis. Among 3p genes, RASSF1A has the highest methylation frequency in epithelial tumors of various locations. For the first time, two different methods (methylation-specific PCR and methylation-sensitive restriction analysis) independently showed that the methylation level of the CpG island in the RASSF1A promoter region significantly correlated with grade and clinical stage of clear cell renal cell carcinoma (RCC). An analysis of 23 3p polymorphic markers in a representative set of 80 RCC cases characterized clinically and histologically revealed that RCC progression significantly correlated with the frequency of allelic imbalances in some critical regions of 3p (LUCA and AP20), but not in 3p as a whole. These data suggest that RCC progression is associated with the methylation of the RASSF1A promoter and, possibly, with structural and functional alterations in other 3p genes. In addition, significant correlation between RASSF1A methylation and allelic losses at the nearby polymorphic marker locus suggests the “two hit” model for the inactivation of this tumor suppressor gene in RCC.     

Retinoblastoma protein family in cell cycle and cancer: A review

Two genes, p107 and Rb2/p130, are strictly related to RB, the most investigated tumor suppressor gene, responsible for susceptibility to retinoblastoma. The products of these three genes, namely pRb, p107, and pRb2/p130 are characterized by a peculiar steric confirmation, called “pocket,” responsible for most of the functional interactions characterizing the activity of these proteins in the homeostasis of the cell cycle. The interest in these genes and proteins springs from their ability to regulate cell cycle processes negatively, being able, for example, to dramatically slow down neoplastic growth. So far, among these genes, only RB is firmly established to act as a tumor suppressor, because its lack-of-function is clearly involved in tumor onset and progression. It has been found deleted or mutated in most retinoblastomas and sarcomas, but its inactivation is likely to play a crucial role in other types of human cancers. The two other members of the family have been discovered more recently and are currently under extensive investigation. We review analogies and differences among the pocket protein family members, in an attempt to understand their functions in normal and cancer cells. © 1996 Wiley-Liss, Inc.     

The two-hit theory hits 50

Few ideas in cancer genetics have been as influential as the “two-hit” theory of tumor suppressors. This idea was introduced in 1971 by Al Knudson in a paper in the Proceedings of the National Academy of Science and forms the basis for our current understanding of the role of mutations in cancer. In this theoretical discussion proposing a genetic basis for retinoblastoma, a childhood cancer of the retina, Knudson posited that these tumors arise from two inactivating mutations, targeting both alleles of a putative tumor suppressor gene. While this work built on earlier proposals that cancers are the result of mutations in more than one gene, it was the first to propose a plausible mechanism by which single genes that are affected by germ-line mutations in heritable cancers could also cause spontaneous, nonheritable tumors when mutated in somatic tissues. Remarkably, Knudson described the existence and properties of a retinoblastoma tumor suppressor gene a full 15 years before the gene was cloned.

Let’s put ourselves, if we may, into the mindset of cancer researchers a half century ago. By the early 1970s, we have come to accept the idea that cancer is a genetic disease resulting from mutations in particular genes. We also know that chromosomal aberrations occur in many cancers, including loss or gain of genetic material, but the identity, and even the existence, of cancer driver genes and how they might operate is entirely unknown to us. Recently reported somatic cell fusion experiments, in which normal human cells have been induced to fuse with malignant rodent cells, suggest that normal cells possess dominant tumor-suppressive properties and that these properties are associated with particular chromosomes (Harris et al., 1969), but the genes that confer such suppression, and how they might do so, are obscure. In addition, there is considerable evidence that that most cancers involve more than a single mutational event (Nordling, 1953; Ashley, 1969). On the other hand, some of our fellow cancer researchers have also shown that acutely transforming retroviruses can rapidly induce cancers in their hosts, suggesting that, at least in avian and mammalian species, single “oncogenes” can transform cells and cause tumors (Huebner and Todaro, 1969). Finally, we know that certain cancer predispositions can be passed down from parent to child, even if the genetic basis for this phenomenon remains uncharacterized.Among the many questions that puzzled our midcentury scientist were: how can these various findings—some of which suggest a multigene cause for cancer and others that suggest a single event—be reconciled? What is the relationship between dominant oncogenes and recessive “anti-oncogenes?” Also, are the genetic mechanisms underlying relatively rare inherited forms of cancer related to the more common forms of this disease, which seem to occur spontaneously?The pediatric cancer retinoblastoma represented a particularly compelling model in which to address this last question. This type of cancer affects retinoblast cells in the developing eye and typically presents in childhood. Curiously, some affected patients were known to develop an early, aggressive, often bilateral form of the disease, and, if they survived, could pass susceptibility to retinoblastoma to their children. Other children developed such tumors later in childhood, never presented with bilateral disease, and did not impart additional risk to their offspring. This cancer drew the attention of Alfred Knudson, at that time a 49-year-old physician studying heritable metabolic disorders. Following a study of patient charts that dated back decades, he suggested in his seminal 1971 National Academy of Science paper that, in both familial and nonfamilial cases of retinoblastoma, the number of mutations required to initiate this tumor was two, that they must occur before retinal cells differentiate, and that the gene or genes affected likely act in a recessive manner (Knudson, 1971). The ideas presented in this paper continue to guide our thinking about cancer genetics to the present day.While the impact and implications of the 1971 paper were profound, the paper itself was profoundly simple. It was four pages long. It had a single author. It neither listed nor required any grant support. It showed no blots (Southern published his eponymous technique in 1975, PCR was more than a decade away, and restriction enzymes were not yet discovered), no sequences (DNA sequencing methods were introduced in 1975), and but one simple figure, showing a straight line and a hyperbolic line on a log scale (Figure 1). In a way, its analytic methods represented a style of science that, while not too uncommon at that time, later fell into relative disfavor as experimental molecular techniques allowed for genes to be isolated, sequenced, mutated, and introduced into cells and animals. By the mid-1970s, gene jockeys were in, and theoretical biologists were out.Open in a separate windowFIGURE 1:The plot from which Knudson proposed the two-hit hypothesis (Knudson, 1971, with the permission of the National Academy of Sciences, USA).Interestingly, Knudson’s statistical approach anticipated much of today’s cancer research literature; that is to say, the work consisted entirely of dataset analysis and mathematical modeling. The numbers being crunched were rather simple by today’s standards: the age of onset of retinoblastoma in pediatric cases, whether these children developed unilateral or bilateral disease, how many tumors were present, and whether these tumors were hereditary or not. The key facts were that the familial cases tended to present at a younger age, were often bilateral, and, in a related point, could arise as multiple independent tumors. He also noted that not everyone who inherited the mutation(s) actually developed tumors; some retinoblastomas skipped a generation. This feature, plus a knowledge of how many cells comprise the retina, suggested that the affected gene(s) was recessive and allowed Knudson to infer a mutational frequency rate per cell division that closely matched previous predictions and was consistent with the observed tumor burden in familial cases.From these data, and unassisted by any form of computer, Knudson used curve-fitting and Poisson statistics to derive an important conclusion: the incidence curve for heritable cases fit a model in which the development of retinoblastoma required not one but two mutational events, or two “hits.” Whether these events were disabling mutations in each of the two alleles of a hypothetical retinoblastoma gene (as indeed proved to be the case) or instead were activating mutations in one allele each of two separate genes, could not be ascertained at that time, though the observation that retinoblastoma cells sometimes lost part of chromosome 13 favored the first interpretation. A decade later, the case for the two-hit theory received crucial experimental support when Cavenee and colleagues applied restriction site polymorphism analysis to retinoblastomas (Cavenee et al., 1983). These studies showed that retinoblastomas commonly display loss of polymorphic restriction sites, consistent with the idea that these tumors involve damage to one allele of an RB gene and subsequent loss of the second copy. The two-hit theory provided an appealing genetic model that could be used to explain both heritable and spontaneous cases of retinoblastoma: the former had one hit in a tumor suppressor gene in the germline and only required one more hit in a somatic retinal cell, whereas the latter required that the first and second mutation to occur in a somatic cell. This model explained why spontaneous cases of retinoblastoma occurred later in life and were never bilateral, as the number of stem cells, the mutation rate, and the amount of time for retinoblasts to terminally differentiate was insufficient for more than one tumor to initiate. The result of these analyses led to a clear prediction regarding the existence and properties of tumor suppressor genes, predictions that have largely withstood the test of time.It would be more than a decade before the first “two-hit” gene, RB, was mapped, isolated, and sequenced (Friend et al., 1986; Lee et al., 1987) and even longer before its biochemical role in regulating cell proliferation was understood in any detail. However, in the meanwhile, dozens of other tumor suppressor genes were characterized, most governed by the rules laid out by Knudson in his 1971 paper.Looking back from a space of 50 years, the 1971 work profoundly reoriented our thinking about cancer genetics in a way that few other single works have done. Importantly, it led to testable predictions that were later—in some cases, much later—proved true. That is not to say, however, that the two-hit theory itself has not evolved. For example, Knudson himself was the one of the first to recognize the possibility that haploinsufficiency (i.e., a one-hit scenario) could alter cellular behavior in ways that contributed to tumorigenesis even in the absence of a second hit. In fact, he spent the last decade of his career studying such effects in cells derived from cancer-prone families (Berger et al., 2011; Peri et al., 2017). Haploinsufficiency was first experimentally verified in mouse models of the Cdkn2a (p27^kip1) tumor suppressor. Mice lacking one allele of Cdkn1b were larger than their littermates, but smaller than those lacking both alleles. Crucially, the heterozygous mice were more prone to tumorigenesis when treated with various mutagens or when bred to oncogene expressing mice (Fero et al., 1998). Many other examples of haploinsufficiency were subsequently described. In this respect, Knudson’s initial two-hit theory was perhaps too parsimonious in its division of tumor suppressor genes into recessive and dominant categories. Most of the proteins encoded by tumor suppressor genes might more aptly be considered as rheostats than as on/off switches: gene dosage matters, and rigid threshold effects are not always seen. To add to the complexity, over the past decades it has become clear that mutations in tumor suppressor genes can also result in dominant-negative or even neomorphic functions, in which the mutant protein carries out functions that are different than those performed the wild-type form (Takiar et al., 2017). To extend our light-switch analogy, the key feature of neomorphic tumor suppressor proteins isn’t whether they act as rheostats or on/off switches, but whether they turn on the stereo instead of the lights. To make matters even more interesting, certain tumor suppressors are suppressors only in particular contexts; that is, depending, as it were, on the time of day and the particulars of the room they’re in, they can act either as on or as off switches. For example, Notch, a central mediator of cell-to-cell signaling, is endowed with both tumor suppressor and tumor-promoting activities that are highly cell and context dependent (Dotto, 2008). Several other tumor suppressor genes display a similar duality (Datta et al., 2020).Another key modification of the two-hit theory is that, despite the simplicity and enduring appeal of the number “two” in its title, the theory applies best to tumor initiation, not necessarily to tumor growth and development. In fact, even in retinoblastoma, it quickly became apparent that two hits are not enough to cause full-blown cancer, and additional “third” hits are required. That is to say, RB1 inactivation is necessary for retinoblastoma tumor initiation but not sufficient for full malignant transformation (Wang et al., 1994; Sellers and Kaelin, 1997).The mapping, cloning, and characterization of additional tumor suppressor genes enabled Kinzler and Vogelstein to propose that these genes fell into at least two general classes: gatekeepers and caretakers (Kinzler and Vogelstein, 1997). The former represented most of the classical tumor suppressor genes, including APC, NF1, NF2, RB1, TSC1/2, VHL, and WT1. These gatekeepers regulate cell division and/or survival through their interaction with elements of signal transduction pathways, and their loss directly initiates growth of the incipient tumor. In contrast, the caretakers, such as ATM, BRCA1 and BRCA2, and FANCA, are involved in maintaining genome integrity through their actions in various aspects of DNA unwinding and repair. In this model, mutational inactivation of such caretaker genes leads to genetic instabilities, increasing the number of mutations of all genes, inactivating gatekeepers and activating oncogenes.In the intervening half century since the initial Knudson paper appeared, the range and variety of mechanisms for tumor suppressor gene inactivation has been more completely defined, incorporating epigenetic as well as genetic events. RB1 itself provides a good example, as silencing of expression of this gene by methylation of CpG islands in its promoter has been noted in sporadic cases (Sakai et al., 1991; Ohtani-Fujita et al., 1993; Greger et al., 1994). In these cases, an epigenetic mechanism of gene inactivation was supported by the lack of mutations in the RB gene sequence. A similar phenomenon has been reported for the VHL gene in spontaneous clear-cell renal carcinoma (Herman et al., 1994) as well as other tumor suppressor genes.Interestingly, at about the same time these ideas were being formulated, Knudson’s colleague at the Institute for Cancer Research (now the Fox Chase Cancer Center), Beatrice Mintz, was busy demonstrating that the cells comprising the tumor cell microenvironment exerted a suppressive effect on cancer cells (Mintz and Illmensee, 1975). In this scenario, loss of a single allele of a tumor suppressor gene in a fibroblast or an immune cell might well impact the growth of an adjacent cancer cell with single or biallelic loss of the same tumor suppressor. A good example of this phenomenon can be seen in one of Knudson’s enduring interests, neurofibromatosis (NF) type 1 syndrome, associated with the tumor suppressor NF1. Here, malignant Schwann cells show biallelic loss of the NF1 gene, just as predicted by proper “Knudsonian” two-hit mechanics, and the surrounding immune cells are hemizygous for NF1 (i.e., have one hit) due to germline mutation. Importantly, these microenvironment cells have to be hemizygous for tumors to develop, as demonstrated by transplantation studies in conditional mouse models (Yang et al., 2008). Such stromal effects have led to the idea that there is a third category of tumor suppressor genes—the landscapers—that predispose to cancer by contributing to a more tumor-conducive stroma (Kinzler and Vogelstein, 1998).Knudson lived to see many of the proteins encoded by tumor suppressor genes functionally linked by virtue of their effects on common signaling pathways that regulate the cell cycle, apoptosis, and protein synthesis. He was particularly interested in determining whether some or all of the tumor suppressor genes that are mutated in phakomatoses—heritable neurocutaneous cancer syndromes that include Cowden’s disease (PTEN), Gorlin’s disease (PTH), juvenile polyposis (SMAD4), Peutz-Jeghers (LKB1), neurofibromatosis type 1 (NF1) and -2 (NF2), tuberous sclerosis (TSC1 and -2), and Von Hippel Lindau (VHL)—could somehow be shown to act in a single pathway, as in fact we now know many of them do. He used to refer to this idea as his “grand unification theory” for tumor suppressor genes.Regarding therapeutics, I think Knudson, whom I knew well as a friend, colleague, and mentor at Fox Chase, would have been disappointed at our relative lack of progress in devising effective treatments for many types of cancers driven by tumor suppressor gene mutations. For example, despite the fact that TP53 is the single most commonly mutated gene in cancer, knowledge of TP53 status has not readily translated into targeted therapies. Part of the reason for this relative lack of progress is obvious: it is much easier to disrupt the action of an oncoprotein than to fix a broken tumor suppressor protein. Direct targeting is not possible if a protein isn’t expressed, and that is the scenario in many tumor cells driven by tumor suppressor gene mutations. Instead, the dominant strategy in this situation has been to target downstream signaling elements, for example, by impeding mitogen-activated protein kinase signaling in NF1-mutant tumors or mTORC1 in TSC-mutant tumors. On the other hand, we have been able to exploit vulnerabilities of cancers with certain caretaker gene mutations, such as PALB2, BRCA1, and BRCA2, as these cells become solely dependent on PARP for DNA repair, rendering them susceptible to small molecule inhibitors of this enzyme. Other “synthetic lethal” strategies have been proposed for various additional tumor suppressor genes (Nijman and Friend, 2013). Finally, given recent advances in gene editing and gene replacement methodologies, it is not unreasonable to think that long before the next 50 years have passed, we will be able to repair damaged tumor suppressor genes in tumor cells and/or replace them with undamaged alleles. If so, we will have come full circle, using a genetic cure for a genetic disease.     

DNA methyltransferase inhibition may limit cancer cell growth by disrupting ribosome biogenesis

Mutations in the pattern of CpG methylation imprinting of the human genome have been correlated with a number of diseases including cancer. In particular, aberrant imprinting of tumor suppressor genes by gain of CpG methylation has been observed in many cancers and thus represents an important alternative pathway to gene mutation and tumor progression. Inhibitors of DNA methylation display therapeutic effects in the treatment of certain cancers and it has been assumed that these effects are due to the reversal of mutant gene imprinting. However, significant reactivation of imprinted tumor suppressor genes is rarely observed in vivo following treatment with DNA methylation inhibitors. A recent study revealed an unexpected requirement for CpG methylation in the synthesis and assembly of the ribosome, an essential function for cell growth and proliferation. As such, the data provide an unforeseen explanation of the action of DNA methylation inhibitors in restricting cancer cell growth.Key words: DNA methylation, meCpG, DNA methyltransferase-inhibition, DNMT1^-/-, DNMT3b^-/-, aza-deoxycytidine, gene silencing, ribosome biogenesis, cancer therapy     

Human breast and colon cancers exhibit alterations of DNA methylation patterns at several DNA segments on chromosomes 11p and 17p

In breast and colon adenocarcinomas methylation patterns at CCGG sites of several loci located on the short arm of chromosome 11 were determined by Southern blot analysis. Results obtained indicate that all tumor samples (20/20) exihibit DNA methylation changes when compared to their normal counterparts. In colon tumors,i-globin gene is usually hypomethylated (9/10), whereas Ha-ras gene, which is located in the same region, retains an unmodified DNA methylation pattern. Hypomethylation of parathyroid hormone (5/10) and catalase genes (4/10) are also frequently detected in colon tumor specimens. For the catalase gene the region around exon is the only one which is affected by these changes. In breast adenocacinoma, modifications of the methylation patterns are less frequently observed. However, hypomethylation of the i-globin gene is a very common event in these tumors (8/10), and it is also detected (2/2) in lobular carcinoma in situ which is an early step in breast tumorgenesis. In addition, hypermethylation of a CpG island is also observed at the locus 17p13.3 in both colon (5/5) and breast (4/9) adenocarcinomas. In the tumoral tissues which containj a limited amount of CpG. Some of these alterations seen, therefore, to be tumor and sequence specific.     

Pathway Implications of Aberrant Global Methylation in Adrenocortical Cancer

Context

Adrenocortical carcinomas (ACC) are a rare tumor type with a poor five-year survival rate and limited treatment options.

Objective

Understanding of the molecular pathogenesis of this disease has been aided by genomic analyses highlighting alterations in TP53, WNT, and IGF signaling pathways. Further elucidation is needed to reveal therapeutically actionable targets in ACC.

Design

In this study, global DNA methylation levels were assessed by the Infinium HumanMethylation450 BeadChip Array on 18 ACC tumors and 6 normal adrenal tissues. A new, non-linear correlation approach, the discretization method, assessed the relationship between DNA methylation/gene expression across ACC tumors.

Results

This correlation analysis revealed epigenetic regulation of genes known to modulate TP53, WNT, and IGF signaling, as well as silencing of the tumor suppressor MARCKS, previously unreported in ACC.

Conclusions

DNA methylation may regulate genes known to play a role in ACC pathogenesis as well as known tumor suppressors.     

The re-expression of the epigenetically silenced e-cadherin gene by a polyamine analogue lysine-specific demethylase-1 (LSD1) inhibitor in human acute myeloid leukemia cell lines

Aberrant epigenetic silencing of tumor suppressor genes is a common feature observed during the transformation process of many cancers, including those of hematologic origin. Histone modifications, including acetylation, phosphorylation, and methylation, collaborate with DNA CpG island methylation to regulate gene expression. The dynamic process of histone methylation is the latest of these epigenetic modifications to be described, and the identification and characterization of LSD1 as a demethylase of lysine 4 of histone H3 (H3K4) has confirmed that both the enzyme and the modified histone play important roles as regulators of gene expression. LSD1 activity contributes to the suppression of gene expression by demethylating promoter-region mono- and dimethyl-H3K4 histone marks that are associated with active gene expression. As most post-translational modifications are reversible, the enzymes involved in the modification of histones have become targets for chemotherapeutic intervention. In this study, we examined the effects of the polyamine analogue LSD1 inhibitor 2d (1,15-bis{N ⁵-[3,3-(diphenyl)propyl]-N ¹-biguanido}-4,12-diazapentadecane) in human acute myeloid leukemia (AML) cell lines. In each line studied, 2d evoked cytotoxicity and inhibited LSD1 activity, as evidenced by increases in the global levels of mono- and di-methylated H3K4 proteins. Global increases in other chromatin modifications were also observed following exposure to 2d, suggesting a broad response to this compound with respect to chromatin regulation. On a gene-specific level, treatment with 2d resulted in the re-expression of e-cadherin, a tumor suppressor gene frequently silenced by epigenetic modification in AML. Quantitative chromatin immunoprecipitation analysis of the e-cadherin promoter further confirmed that this re-expression was concurrent with changes in both active and repressive histone marks that were consistent with LSD1 inhibition. As hematologic malignancies have demonstrated promising clinical responses to agents targeting epigenetic silencing, this polyamine analogue LSD1 inhibitor presents an exciting new avenue for the development of novel therapeutic agents for the treatment of AML.     

A reinvestigation of somatic hypermethylation at the PTEN CpG island in cancer cell lines

Background

PTEN is an important tumour suppressor gene that is mutated in Cowden syndrome as well as various sporadic cancers. CpG island hypermethylation is another route to tumour suppressor gene inactivation, however, the literature regarding PTEN hypermethylation in cancer is controversial. Furthermore, investigation of the methylation status of the PTEN CpG island is challenging due to sequence homology with the PTEN pseudogene, PTENP1. PTEN shares a CpG island promoter with another gene known as KLLN. Here we present a thorough reinvestigation of the methylation status of the PTEN CpG island in DNA from colorectal, breast, ovarian, glioma, lung and haematological cancer cell lines.

Results

Using a range of bisulphite-based PCR assays we investigated 6 regions across the PTEN CpG island. We found that regions 1-4 were not methylated in cancer cell lines (0/36). By allelic bisulphite sequencing and pyrosequencing methylation was detected in regions 5 and 6 in colorectal, breast and haematological cancer cell lines. However, methylation detected in this region was associated with the PTENP1 promoter and not the PTEN CpG island.

Conclusions

We show that methylation of the PTEN CpG island is a rare event in cancer cell lines and that apparent methylation most likely originates from homologous regions of the PTENP1 pseudogene promoter. Future studies should utilize assays that reliably discriminate between PTEN and PTENP1 to avoid data misinterpretation.     

Tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns

Background

Aberrant CpG island promoter DNA hypermethylation is frequently observed in cancer and is believed to contribute to tumor progression by silencing the expression of tumor suppressor genes. Previously, we observed that promoter hypermethylation in breast cancer reflects cell lineage rather than tumor progression and occurs at genes that are already repressed in a lineage-specific manner. To investigate the generality of our observation we analyzed the methylation profiles of 1,154 cancers from 7 different tissue types.

Results

We find that 1,009 genes are prone to hypermethylation in these 7 types of cancer. Nearly half of these genes varied in their susceptibility to hypermethylation between different cancer types. We show that the expression status of hypermethylation prone genes in the originator tissue determines their propensity to become hypermethylated in cancer; specifically, genes that are normally repressed in a tissue are prone to hypermethylation in cancers derived from that tissue. We also show that the promoter regions of hypermethylation-prone genes are depleted of repetitive elements and that DNA sequence around the same promoters is evolutionarily conserved. We propose that these two characteristics reflect tissue-specific gene promoter architecture regulating the expression of these hypermethylation prone genes in normal tissues.

Conclusions

As aberrantly hypermethylated genes are already repressed in pre-cancerous tissue, we suggest that their hypermethylation does not directly contribute to cancer development via silencing. Instead aberrant hypermethylation reflects developmental history and the perturbation of epigenetic mechanisms maintaining these repressed promoters in a hypomethylated state in normal cells.     

Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis

Aberrant DNA methylation patterns have been reported in inflamed tissues and may play a role in disease. We studied DNA methylation and gene expression profiles of purified intestinal epithelial cells from ulcerative colitis patients, comparing inflamed and non-inflamed areas of the colon. We identified 577 differentially methylated sites (false discovery rate <0.2) mapping to 210 genes. From gene expression data from the same epithelial cells, we identified 62 differentially expressed genes with increased expression in the presence of inflammation at prostate cancer susceptibility genes PRAC1 and PRAC2. Four genes showed inverse correlation between methylation and gene expression; ROR1, GXYLT2, FOXA2, and, notably, RARB, a gene previously identified as a tumor suppressor in colorectal adenocarcinoma as well as breast, lung and prostate cancer. We highlight targeted and specific patterns of DNA methylation and gene expression in epithelial cells from inflamed colon, while challenging the importance of epithelial cells in the pathogenesis of chronic inflammation.     

<Emphasis Type="Italic">BMP3</Emphasis> promoter hypermethylation in plasma-derived cell-free DNA in colorectal cancer patients

Detecting cfDNA in plasma or serum could serve as a ‘liquid biopsy’, for circulating tumor DNA with aberrant methylation patterns offer a possible method for early detection of several cancers which could avoid the need for tumor tissue biopsies. Bone Morphogenetic Protein 3 (BMP3) was identified as a candidate tumor suppressor gene putatively down-regulated in colorectal cancer (CRC). In this study, we aimed to assess the potential role of BMP3 promoter methylation changes in plasma DNA for detection of colorectal cancerous and precancerous lesions. Plasma DNA samples were extracted from 50 patients with histologically diagnosed polyps or tumor and 50 patients reported negative for polyps or tumors. The procedure consists of bisulfite conversion of the extracted DNA, purification of bis-DNA, and BMP3 methylation status analysis by using the bisulfite specific high resolution melting analysis. This study demonstrated that there was a significantly higher frequency of BMP3 methylated DNA in plasma in patients with polyps versus healthy controls with a sensitivity and specificity of 40 and 94%, respectively. In conclusion, our results demonstrated that BMP3 DNA methylation in plasma had not have sufficient sensitivity and it should be used in combination with other biomarkers for the detection of CRC.     

Methylation of the Promoter Region of the RASSF1A Candidate Tumor Suppressor Gene in Primary Epithelial Tumors

The methylation of the promoter CpG island of the RASSF1A tumor suppressor gene in primary tumors of 172 Muscovites with renal cell carcinoma (RCC), breast cancer (BC), or ovarian epithelial tumors (OET) was assayed by means of methylation-specific PCR (MSP) and PCR-based methylation-sensitive restriction enzyme analysis (MSRA). The MSP, MSRA, and previous bisulfite sequencing data correlated significantly with each other (P 10^–6 for Spearman's rank correlation coefficients). By MSP and MSRA, the respective methylation frequencies of the RASSF1A promoter were 86% (25/29) and 94% (50/53) in RCC, 64% (18/28) and 78% (32/41) in BC, and 59% (17/29) and 73% (33/45) in OET. Methylation-sensitive restriction enzymes (HpaII, HhaI, Bsh1236I, AciI) increased the analysis sensitivity and made it possible to establish the methylation status for 18 CpG dinucleotides of the RASSF1A promoter region. With the MSRA data, the density of methylation of the CpG island was estimated at 72% in RCC, 63% in BC, and 58% in OET (the product of the number of CpG dinucleotides and the number of specimens with RASSF1A methylation was taken as 100%). Methylation of the RASSF1A promoter region was observed in 11–35% of the DNA specimens from the histologically normal tissue adjacent to the tumor but not in the peripheral blood DNA of 15 healthy subjects. The RASSF1A methylation frequency showed no significant correlation with the stage, grade, and metastatic potential of the tumor. On the other hand, epigenetic modification of RASSF1A was considerably more frequent than hemizygous or homozygous deletions from the RASSF1A region. These results testify that methylation of the RASSF1A promoter region takes place early in carcinogenesis and is a major mechanism inactivating RASSF1A in epithelial tumors.