Spiral plasmonic nanoantennas as circular polarization transmission filters.

We present simulation and experimental results for easily fabricated spiral plasmonic antenna analogues providing circular polarization selectivity. One circular polarization state is concentrated and transmitted through a subwavelength aperture, while the opposite circular state is blocked. The spectral bandwidth, efficiency, and extinction ratios are tunable through geometric parameters. Integration of such structures onto a focal plane array in conjunction with linear micropolarizers enables complete Stokes vector imaging, that, until now, has been difficult to achieve. An array of these structures forms a plasmonic metamaterial that exhibits high circular dichroism.

Deletion of p53 in human mammary epithelial cells causes chromosomal instability and altered therapeutic response.

The TP53 tumor suppressor gene is the most commonly mutated gene in human cancers. To evaluate the biological and clinical relevance of p53 loss, human somatic cell gene targeting was used to delete the TP53 gene in the non-tumorigenic epithelial cell line, MCF-10A. In all four p53-/- clones generated, cells acquired the capability for epidermal growth factor-independent growth and were defective in appropriate downstream signaling and cell cycle checkpoints in response to DNA damage. Interestingly, p53 loss induced chromosomal instability leading to features of transformation and the selection of clones with varying phenotypes. For example, p53-deficient clones were heterogeneous in their capacity for anchorage-independent growth and invasion. In addition, and of clinical importance, the cohort of p53-null clones showed sensitivity to chemotherapeutic interventions that varied depending not only on the type of chemotherapeutic agent, but also on the treatment schedule. In conclusion, deletion of the TP53 gene from MCF-10A cells eliminated p53 functions, as well as produced p53-/- clones with varying phenotypes possibly stemming from the distinct chromosomal changes observed. Such a model system will be useful to further understand the cancer-specific phenotypic changes that accompany p53 loss, as well as help to provide future treatment strategies for human malignancies that harbor aberrant p53.

Short communication: Effect of estrogen supplemented at dry-off on temporal changes in concentrations of lactose in blood plasma of Holstein cows.

The objective was to determine the effect of supplemental estrogen (estradiol cypionate, ECP) at dry-off on temporal changes in concentrations of lactose in blood plasma of Holstein cows as an indicator of rate of mammary involution. Thirty-two Holstein cows (8/group) were assigned randomly to 4 treatment groups: 30-d dry, 30-d dry + ECP, 60-d dry, and 60-d dry + ECP. A single injection (7.5 mL) of cottonseed oil (30- and 60-d dry) or ECP (15 mg) in oil (30- and 60-d dry + ECP) was administered intramuscularly at dry-off. Blood samples were collected from the coccygeal vein of all cows 24 h before dry-off and at dry-off, and then 8 samples were collected throughout the subsequent 48 h to monitor concentrations of lactose in blood plasma. No significant effects of ECP on the overall mean concentrations of lactose were detected. Concentrations of lactose increased and were greatest in blood collected 20 h (520.4 +/- 54.1, 268.1 +/- 48.2, 345.0 +/- 52.3, 418.4 +/- 49.8 microM, for the 4 treatment groups respective to the order listed above) after supplemental ECP and final milk removal. At 40 h, concentrations approached those observed 24 h before dry-off (140.5 +/- 52.1, 57.6 +/- 47.1, 90.1 +/- 51.4, 61.2 +/- 48.4 microM, respectively). Concentrations of lactose at 20 h were positively correlated with milk yield of cows at dry-off. Similar temporal profiles of lactose in blood plasma of cows supplemented or not with ECP indicated that ECP at dry-off did not markedly alter the course of tight junction leakage that typically occurs in mammary epithelial tissue during progressive early involution when milk removal is discontinued.

Persistent mismatch repair deficiency following targeted correction of hMLH1.

The use of gene therapy to correct mutated or lost gene function for the treatment of human cancers has been an active, yet problematic area of biomedical research. Many technical difficulties, including efficient tissue-specific delivery, integration site specificity and general toxicity, are being addressed. Little is known, however, about the genetic and phenotypic stability that accompanies a successful gene-specific targeting event in a cancer cell. This question was addressed following the creation of a colon cancer cell line in which a mutated hMLH1 gene was corrected via targeted homologous recombination. This correction resulted in the expression of wild-type hMLH1 protein, restoration of the hPMS2 protein and mismatch repair (MMR) proficiency. One of two hMLH1-corrected clones, however, was found to retain defects in MMR activity. These cells continued to express the corrected hMLH1 protein, but had lost expression of another MMR protein, hMSH6. DNA sequence analysis of the hMSH6 gene revealed biallelic expansions of a cytosine repeat region in exon 5 that result in frameshifts leading to premature stop codons. These findings suggest that, similar to acquired drug resistance, the presence of genetically heterogeneous cancer cell populations or acquisition of compensatory mutations can result in 'resistance' to gene replacement therapy.

Interleukin-1 alpha mediates the growth proliferative effects of transforming growth factor-beta in p21 null MCF-10A human mammary epithelial cells.

Transforming growth factor-beta type 1 (TGF-beta) has been implicated as both a tumor suppressor and a tumor promoter in many solid epithelial cancers. We have previously demonstrated that the cyclin dependent kinase (CDK) inhibitor p21 acts as a molecular switch in determining a growth inhibitory versus growth proliferative response to TGF-beta in the spontaneously immortalized human mammary epithelial cell line MCF-10A. We now demonstrate that this proliferative effect of TGF-beta is mediated through the proinflammatory cytokine, interleukin-1alpha (IL-1alpha). Using gene expression array analysis, we identified IL-1alpha as a cytokine specifically upregulated only in cells lacking p21 and only upon TGF-beta stimulation. Cell proliferation assays verified that recombinant IL-1alpha was capable of inducing a growth proliferative response in p21 null MCF-10A cells, while neutralizing antibodies against IL-1alpha prevented the growth proliferative effects of TGF-beta. Mechanistically, both the CDK and proliferating cell nuclear antigen (PCNA) inhibitory functions of p21 were responsible for preventing TGF-beta induced cell proliferation, but only PCNA inhibition by p21 regulated IL-1alpha gene expression. These studies demonstrate a novel role for IL-1alpha in mediating a proliferative response to TGF-beta signaling, and suggest that therapies directed against IL-1alpha could abate the growth proliferative effects of TGF-beta without compromising its tumor suppressive function.

Mutation of the PIK3CA oncogene in human cancers.

It is now well established that cancer is a genetic disease and that somatic mutations of oncogenes and tumour suppressor genes are the initiators of the carcinogenic process. The phosphatidylinositol 3-kinase signalling pathway has previously been implicated in tumorigenesis, and evidence over the past year suggests a pivotal role for the phosphatidylinositol 3-kinase catalytic subunit, PIK3CA, in human cancers. In this review, we analyse recent reports describing PIK3CA mutations in a variety of human malignancies, and discuss their possible implications for diagnosis and therapy.

Milk production from Holstein half udders after concurrent thirty- and seventy-day dry periods.

The objective of this study was to use a within-cow, half-udder model to compare the effect of cessation of milk removal from mammary quarters within respective half udders at either 30 or 70 d before expected calving date (ECD) on the ability of the half udders to subsequently produce milk. Pregnant Holstein cows were assigned to control (n = 14) or treatment (TRT, n = 26) groups. All mammary quarters in the udder of cows in the control group had 70-d (68 +/- 9 d) dry periods, whereas in each cow of the TRT group, 1 randomly selected half udder was dried at 70 d before ECD and the other half udder continued to be milked twice daily until dried at 30 d before ECD. From 80 through 70 d before ECD, amounts of milk produced by the left and right half udders of cows in the TRT group were measured at the first-shift milking. No differences were detected in the actual or relative amounts of milk produced by the left (3.46 +/- 0.2 kg; 48.8 +/- 1.0%) and the right (3.63 +/- 0.2 kg; 51.2 +/- 1.0%) half udders. Furthermore, the actual and relative amounts of milk produced by the half udders (n = 12 left, 14 right) subsequently dry for 67 +/- 7 d (3.56 +/- 0.2 kg; 50.2 +/- 1.0%) and the half udders (n = 14 left, 12 right) subsequently dry for 27 +/- 7 d (3.54 +/- 0.2 kg; 49.8 +/- 1.0%) did not differ before they were dried. However, from 3 to 100 d of the subsequent lactation, the 30-d dry half udders produced 18.9% less milk than the 70-d dry half udders (16.3 vs. 20.1 +/- 1.0 kg/d). In addition, relative amounts of total-udder milk produced by the 30- and 70-d dry half udders in the same cow differed (44.9 vs. 55.1 +/- 0.2%, respectively). Cows in the control group produced more milk than cows in the TRT group through 80 DIM (39.5 vs. 35.2 +/- 0.6 kg/d), but not from 3 through 150 DIM (39.0 vs. 36.2 +/- 1.6 kg/d). Thus, half udders that produced the same actual and relative amounts of milk before being dried did not do so when given a 30-d dry period instead of a 70-d dry period. When compared with the pre-dry value (49.8%), the relative contribution of half udders dry for 30 d to the total milk yield during the first 100 DIM was decreased by 9.8%.

Invited review: bovine studies on optimal lengths of dry periods.

Milk production per cow has increased as a result of progressive changes in the genetics and management of the dairy animal population. A management constant during many decades of progress has been the widely adopted dry period length of 51 to 60 d. The scientific basis for that industry standard was examined to assess its validity as the appropriate standard for the modern dairy industry. If subsequent milk yields can be sustained fully after dry periods that are shorter than the current standard, then considerable milk is being forfeited by retaining longer dry periods. Conversely, failure to allow any dry period will result in a significant decrease in subsequent milk synthesis and secretion. Most studies to determine the minimum length of dry period required have involved retrospective analyses of observational data. Only five experiments have been reported in which dairy cows were assigned, at random, to planned 30- and 60-d dry periods. Estimates of the change in subsequent milk production when days dry were decreased from 50 to 57 d to 30 to 34 d ranged from a 10% decrease to a 1% increase. However, lower yields after shorter dry periods may be partially offset by greater milk yields in the previous lactation if such cows are milked 3 to 4 wk longer. Environmental factors that influence milk production as well as the biological processes that occur within the mammary gland during the nonlactating period must be considered when dry period lengths are compared. Importantly, additional animal trials that specifically assign cows randomly to the dry period lengths to be evaluated are needed to determine optimal dry period lengths for modern dairy cows in differing management scenarios.

Milk production and feed intake of Holstein cows given short (30-d) or normal (60-d) dry periods.

Eighty-four Holstein cows were utilized to evaluate effects of dry period (60 d vs. 30 d), with or without estradiol cypionate (ECP) injections to accelerate mammary involution, on prepartum and postpartum dry matter intake (DMI), body weight (BW), body condition score (BCS), and subsequent milk yield (MY). Treatments were arranged in a 3 x 2 x 2 factorial design that included dry period (30 d dry, 30 d dry + ECP, and 60 d dry), prepartum and postpartum bovine somatotropin (bST; 10.2 mg/d), and prepartum anionic or cationic diets. To accelerate mammary involution, ECP (15 mg) was injected intramuscularly at dry-off. No interaction of bST or prepartum diet with dry period length was detected on BW, BCS, or MY. No significant effects of dry period length on prepartum DMI, BW, or BCS were detected. Cows with shorter dry periods maintained postpartum BCS better and tended to have greater DMI immediately postpartum. Mean daily yields of milk for dry period groups did not differ during overall lactation period (1 to 21 wk). Injection of ECP at the onset of the 30-d dry period did not affect MY. No significant differences due to dry period length were detected for milk, 3.5% FCM, or SCM yields during first 10 wk of lactation. Data indicated that a short dry period protocol can be used as a management tool with no loss in the subsequent milk production of dairy cows.

Milk production of dairy cows treated with estrogen at the onset of a short dry period.

The objective was to determine whether the use of estradiol-17beta (E2) at the initiation of short dry periods prevented an anticipated decline in milk production in the subsequent lactation. Lactating Holstein cows (n = 66) were dried at either 60 or 30 d before expected calving. Treatments in a 2 x 2 factorial arrangement included: D60 (n = 19, 60-d dry, no E2), D60 + E2 (n = 18, 60-d dry, E2), D30 (n = 15, 30-d dry, no E2), and D30 + E2 (n = 14, 30-d dry, E2). To accelerate mammary involution, estradiol-17beta (15 mg in 4 ml of ethanol) was injected subcutaneously daily for 4 d beginning 30 d before expected calving. Parturitions occurred between November 1995, and March 1996. Actual days dry for respective treatments were 57.3, 60.6, 33.9, and 33.8 +/- 1.7 d. Onset of parturition, calving difficulty, and cow health were not affected by E2. Actual 305-d milk yields for the lactation completed immediately before the experimental dry period were 10,318, 10,635, 10,127, and 10,447 +/- 334 kg, respectively; and were 9942, 9887, 9669, and 10,172 +/- 387 kg, respectively, for the lactation immediately following treatment. Respective pre- and posttreatment mature equivalent 305-d yields were 9574, 9861, 9812, and 9724 +/- 297 kg; 8987, 8843, 9126, and 9008 +/- 294 kg. Milk yields did not differ across treatments. Cows with a 34-d dry period were as productive as cows with a 59-d dry period. Estradiol-17beta had no effect, but perhaps should be evaluated with dry periods shorter than 34 d.

DNA methylation, chromatin inheritance, and cancer.

Cancer is a process driven by the accumulation of abnormalities in gene function. While many of these changes are genetic, epigenetically mediated changes in gene expression are being increasingly appreciated. This latter process emphasizes the need to understand two key components of heritable, but reversible, modulation of gene promoter function that are closely tied to one another - formation of chromatin which modulates transcription and establishing patterns of DNA methylation. The link lies first in the recruitment to methylated cytosines of a family of methyl-CpG binding domain proteins (MBDs), which are direct transcriptional repressors and can complex with transcriptional corepressors including histone deacetylases (HDACs). Additionally, the proteins that catalyze DNA methylation, DNA methyltransferases (DNMTs), also directly repress transcription and associate with HDACs. Regulation of these above chromatin-DNA methylation interactions as a function of DNA replication timing is emerging as a key event in the inheritance of transcriptionally repressed domains of the genome. Importantly, synergy between HDAC activity and DNA methylation is operative for a key epigenetic abnormality in cancer cells, transcriptional silencing of tumor suppressor genes. This change has now been recognized for genes that are essential for normal regulation of virtually every major cell function including cell growth, differentiation, apoptosis, DNA repair, and cell-cell, cell-substratum interaction. Understanding the molecular determinants of both normal and abnormal patterns of chromatin formation and DNA methylation thus holds great promise for our understanding of cancer and for means to better diagnose, prevent, and treat this disease.

Dnmt3a and Dnmt3b are transcriptional repressors that exhibit unique localization properties to heterochromatin.

We demonstrate that the recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, like DNMT1, repress transcription in a methylation-independent manner. Dnmt3a and Dnmt3b repress transcription primarily through a plant homeodomain-like motif that is shared with the ATRX protein but is not present in DNMT1. Unlike DNMT1, which localizes to replication foci during S-phase in murine embryonic fibroblasts, Dnmt3a co-localizes with heterochromatin protein 1 alpha (HP1 alpha) and methyl-CpG binding proteins throughout the cell cycle to late-replicating pericentromeric heterochromatin. In contrast to Dnmt3a, Dnmt3b remained diffuse in the nucleus of embryonic fibroblasts at all cell cycle stages. However, Dnmt3a and Dnmt3b co-localize to these pericentromeric heterochromatin regions in murine embryonic stem cells. This finding is important to the fact that mutations in DNMT3B are found in the developmental syndrome, ICF (immunodeficiency, centromeric heterochromatin instability, and facial anomalies), which involves extensive loss of methylation from pericentromeric regions. The localization of Dnmt3a and Dnmt3b was unaffected in Dnmt1 null embryonic stem cells, which lose the majority of methylation at pericentromeric major satellite repeats, suggesting that these enzymes are not dependent upon preexisting methylation for their targeting. DNMT1 is then positioned to reestablish transcriptionally repressive chromatin as cells replicate, while Dnmt3a and Dnmt3b may help to establish such chromatin in late S-phase and maintain this repressive heterochromatin throughout the cell cycle in a developmentally and/or cell type manner.

Aberrant patterns of DNA methylation, chromatin formation and gene expression in cancer.

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.

Mechanism of topical glucocorticoid treatment of hay fever: IL-5 and eosinophil activation during natural allergen exposure are suppressed, but IL-4, IL-6, and IgE antibody production are unaffected.

BACKGROUND: Allergic rhinitis is traditionally defined as an IgE- and mast cell-mediated hypersensitivity reaction. Allergen challenge models suggest that cytokines and eosinophil mediators may also play roles. However, the causal relationship among inflammatory cells, their products, and patients' symptoms during natural allergen exposure has not been established. OBJECTIVE: We sought to elucidate the mechanisms of seasonal allergic rhinitis and the beneficial effects of topical glucocorticoids. METHODS: Thirty patients with ragweed-induced hay fever and a strongly positive serologic test response for ragweed IgE antibody received budesonide nasal spray or placebo in a randomized, parallel, double-blind study. Nasal wash fluids and sera were collected before and during the hay fever season. The levels of inflammatory mediators and allergen-specific immunoglobulins were measured by immunoassay. The activation markers on blood eosinophils were quantitated by flow cytometry. RESULTS: Compared with placebo-treated patients, budesonide-treated patients had strikingly reduced symptoms. In the placebo group, nasal symptoms correlated with nasal lavage fluid eosinophil-derived neurotoxin and IL-5 levels. At the season peak, the budesonide-treated group had significantly lower nasal fluid eosinophil-derived neurotoxin, IL-5, and soluble intracellular adhesion molecule-1 levels. In the treated group eosinophil expression of CD11b was suppressed at the season peak. In contrast, levels of IL-4 and IL-6 in nasal fluid and the seasonal increases in serum ragweed-specific IgE and nasal fluid IgA antibodies did not differ between groups. CONCLUSION: Eosinophilic inflammation plays a critical role in seasonal allergic rhinitis symptoms. One of the therapeutic effects of glucocorticoids is to suppress this inflammation.

Decreased expression of the gut-enriched Krüppel-like factor gene in intestinal adenomas of multiple intestinal neoplasia mice and in colonic adenomas of familial adenomatous polyposis patients.

Gut-enriched Krüppel-like factor (GKLF) is a zinc finger-containing transcription factor, the expression of which is associated with growth arrest. We compared Gklf expression in intestinal and colonic adenomas to normal mucosa in multiple intestinal neoplasia (Min) mice and familial adenomatous polyposis (FAP) patients, respectively, using semi-quantitative RT-PCR. In Min mice, the level of Gklf transcript is highest in normal-appearing intestinal tissues and decreases as the size of the adenoma increases. In FAP patients, the level of GKLF transcript is lower in adenomas compared to paired normal-appearing mucosa from the same patient or normal colonic mucosa from control individuals without FAP. The possibility of DNA methylation as a cause for the decreased expression of Gklf in adenomas of Min mice was investigated by methylation-specific PCR. Results indicate that the Gklf gene is not methylated in either normal or tumorous tissues. The findings of our study are therefore consistent with the potential role of GKLF as a negative growth regulator of gut epithelial cells.

DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci.

DNA methylation can contribute to transcriptional silencing through several transcriptionally repressive complexes, which include methyl-CpG binding domain proteins (MBDs) and histone deacetylases (HDACs). We show here that the chief enzyme that maintains mammalian DNA methylation, DNMT1, can also establish a repressive transcription complex. The non-catalytic amino terminus of DNMT1 binds to HDAC2 and a new protein, DMAP1 (for DNMT1 associated protein), and can mediate transcriptional repression. DMAP1 has intrinsic transcription repressive activity, and binds to the transcriptional co-repressor TSG101. DMAP1 is targeted to replication foci through interaction with the far N terminus of DNMT1 throughout S phase, whereas HDAC2 joins DNMT1 and DMAP1 only during late S phase, providing a platform for how histones may become deacetylated in heterochromatin following replication. Thus, DNMT1 not only maintains DNA methylation, but also may directly target, in a heritable manner, transcriptionally repressive chromatin to the genome during DNA replication.

HMO physicians' use of referrals.

Clinical uncertainty is a source of variation in medical decision-making as well as a source of work-related stress. Increasing enrollment in organized health care systems has intensified interest in understanding referral utilization as well as issues such as physician dissatisfaction and burnout. We examined whether primary care physicians' affective reactions to uncertainty and their job characteristics were associated with use of referrals and burnout. Data came from mail surveys of primary care physicians practicing in two large group model health maintenance organizations (HMOs) in the USA. Consistent with past research, we found that younger physicians had higher referral rates than older physicians, and that general internists had higher rates than either family practitioners or pediatricians. Greater stress from uncertainty increased referrals and referrals were negatively correlated with heavier work demands (patient visits per hour). Greater stress from uncertainty, perceived workload (too high) and a sense of loss of control over the practice environment were associated with higher levels of burnout.

Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) antagonizes matrix metalloproteinase activity and can suppress tumor growth, angiogenesis, invasion, and metastasis. Loss of TIMP-3 has been related to the acquisition of tumorigenesis. Herein, we show that TIMP-3 is silenced in association with aberrant promoter-region methylation in cell lines derived from human cancers. TIMP-3 expression was restored after 5-aza-2'deoxycytidine-mediated demethylation of the TIMP-3 proximal promoter region. Genomic bisulfite sequencing revealed that TIMP-3 silencing was related to the overall density of methylation and that discrete regions within the TIMP-3 CpG island may be important for the silencing of this gene. Aberrant methylation of TIMP-3 occurred in primary cancers of the kidney, brain, colon, breast, and lung, but not in any of 41 normal tissue samples. The most frequent TIMP-3 methylation was found in renal cancers, which originate in the tissue that normally expresses the highest TIMP-3 levels. This methylation correlated with a lack of detectable TIMP-3 protein in these tumors. Together, these data show that methylation-associated inactivation of TIMP-3 is frequent in many human tumors.

Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer.

Densely methylated DNA associates with transcriptionally repressive chromatin characterized by the presence of underacetylated histones. Recently, these two epigenetic processes have been dynamically linked. The methyl-CpG-binding protein MeCP2 appears to reside in a complex with histone deacetylase activity. MeCP2 can mediate formation of transcriptionally repressive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone deacetylase. Little is known, however, about the relative roles of methylation and histone deacetylase activity in the stable inhibition of transcription on densely methylated endogenous promoters, such as those for silenced alleles of imprinted genes, genes on the female inactive X chromosome and tumour-suppressor genes inactivated in cancer cells. We show here that the hypermethylated genes MLH1, TIMP3 (TIMP3), CDKN2B (INK4B, p15) and CDKN2A (INK4, p16) cannot be transcriptionally reactivated with TSA alone in tumour cells in which we have shown that TSA alone can upregulate the expression of non-methylated genes. Following minimal demethylation and slight gene reactivation in the presence of low dose 5-aza-2'deoxycytidine (5Aza-dC), however, TSA treatment results in robust re-expression of each gene. TSA does not contribute to demethylation of the genes, and none of the treatments alter the chromatin structure associated with the hypermethylated promoters. Thus, although DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.