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DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA–mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based “epigenetic unmasking” in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries. (Mol Cancer Res 2008;6(10):1567–74)

Supplementary Tables S1-S2 from Genome-Wide Analysis in a Murine Dnmt1 Knockdown Model Identifies Epigenetically Silenced Genes in Primary Human Pituitary Tumors

Several recent reports have described associations between gestational diabetes (GDM) and changes to the epigenomic landscape where the DNA samples were derived from either cord or placental sources. We employed genome-wide 450Karray analysis to determine changes to the epigenome in a unique cohort of maternal blood DNA from 11 pregnant women prior to GDM development relative to matched controls. Hierarchical clustering segregated the samples into two distinct clusters comprising GDM and healthy pregnancies. Screening identified 100 CpGs with a mean β-value difference of ≥0.2 between cases and controls. Using stringent criteria, 5 CpGs (within COPS8, PIK3R5, HAAO, CCDC124, and C5orf34 genes) demonstrated potentials to be clinical biomarkers as revealed by differential methylation in 8 of 11 women who developed GDM relative to matched controls. We identified, for the first time, maternal methylation changes prior to the onset of GDM that may prove useful as biomarkers for early therapeutic intervention.

High-grade non-muscle invasive bladder cancer (HG-NMIBC) is a clinically unpredictable disease with greater risks of recurrence and progression relative to their low-intermediate-grade counterparts. The molecular events, including those affecting the epigenome, that characterize this disease entity in the context of tumor development, recurrence, and progression, are incompletely understood. We therefore interrogated genome-wide DNA methylation using HumanMethylation450 BeadChip arrays in 21 primary HG-NMIBC tumors relative to normal bladder controls. Using strict inclusion-exclusion criteria we identified 1,057 hypermethylated CpGs within gene promoter-associated CpG islands, representing 256 genes. We validated the array data by bisulphite pyrosequencing and examined 25 array-identified candidate genes in an independent cohort of 30 HG-NMIBC and 18 low-intermediate-grade NMIBC. These analyses revealed significantly higher methylation frequencies in high-grade tumors relative to low-intermediate-grade tumors for the ATP5G2, IRX1 and VAX2 genes (P

Aim: Although aberrant DNA methylation has been described in rheumatoid arthritis (RA), no studies have interrogated this epigenetic modification in early disease. Following recent investigations of T and B lymphocytes in established disease, we now characterize in these cell populations genome-wide DNA methylation in treatment-naive patients with early RA. Patients & methods: HumanMethylation450 BeadChips were used to examine genome-wide DNA methylation in lymphocyte populations from 23 early RA patients and 11 healthy individuals. Results: Approximately 2000 CpGs in each cell type were differentially methylated in early RA. Clustering analysis identified a novel methylation signature in each cell type (150 sites in T lymphocytes, 113 sites in B lymphocytes) that clustered all patients separately from controls. A subset of sites differentially methylated in early RA displayed similar changes in established disease. Conclusion: Treatment-naive early RA patients display novel disease-specific DNA methylation aberrations, supporting a potential role for these changes in the development of RA.

// Marije J. Veenstra 1 , Peter M. van Koetsveld 1 , Fadime Dogan 1 , William E. Farrell 2 , Richard A. Feelders 1 , Steven W.J. Lamberts 1 , Wouter W. de Herder 1 , Giovanni Vitale 3, 4 , Leo J. Hofland 1 1 Department of Internal Medicine, Division of Endocrinology, Erasmus MC, Rotterdam, The Netherlands 2 Department Human Disease and Genomics Group, Institute of Science and Technology in Medicine, School of Medicine, Keele University, Keele, United Kingdom 3 Laboratory of Endocrine and Metabolic Research, Istituto Auxologico Italiano IRCCS, Milan, Italy 4 Department of Clinical Sciences and Community Health (DISCCO), University of Milan, Milan, Italy Correspondence to: Leo J. Hofland, email: l.hofland@erasmusmc.nl Keywords: pancreatic neuroendocrine tumor, somatostatin receptor type 2, epigenetics, BON-1, QGP-1 Received: November 19, 2015 Accepted: April 24, 2016 Published: May 19, 2016 ABSTRACT Somatostatin receptors are a pivotal target for treatment of pancreatic neuroendocrine tumors (pNET), either with somatostatin analogues (SSA) or radiolabeled SSA. The highest affinity target for the most commonly used SSA is the somatostatin receptor type 2 ( sst 2 ). An important factor that may complicate treatment efficacy, is the variable number of receptors expressed on pNETs. Gene expression is subject to complex regulation, in which epigenetics has a central role. In this study we explored the possible role of epigenetic modifications in the variations in sst 2 expression levels in two human pNET cell lines, BON-1 and QGP-1. We found upregulation of sst 2 mRNA after treatment with the epidrugs 5-aza-2’-deoxycytidine (5-aza-dC) and valproic acid (VPA), an increased uptake of radiolabeled octreotide, as well as increased sensitivity to the SSA octreotide in functional cAMP inhibition. At epigenetic level we observed low methylation levels of the sst 2 gene promoter region irrespective of expression. Activating histone mark H3K9Ac can be regulated with epidrug treatment, with an angle of effect corresponding to the effect on mRNA expression. Repressive histone mark H3K27me3 is not regulated by either 5-aza-dC or VPA. We conclude that epidrug treatment, in particular with combined 5-aza-dC and VPA treatment, might hold promise for improving and adding to current SSA treatment strategies of patients with pNETs.

Background Epigenetic regulation of immune cell types could be critical for the development and maintenance of autoimmune diseases like Rheumatoid Arthritis (RA). B cells are highly relevant in RA, since patients express autoantibodies and depleting this cell type is a successful therapeutic approach. Epigenetic variation, such as DNA methylation, may mediate the pathogenic activity of B cells. Objectives In this study, we performed an epigenome-wide association study (EWAS) for RA with three different replication cohorts, to identify disease-specific alterations in DNA methylation in B cells. Methods Genomic methylation in isolated B lymphocytes was assayed on the Illumina HumanMethylation450 BeadChip, assaying >450,000 different CpG sites. Differential methylated positions (DMPs) were identified in a discovery cohort using a single-point analysis using logistic regression, as well as a pathway-level analysis using a newly developed permutation-based method. A discovery cohort of 50 RA patients and 75 healthy controls from Spain was used to identify the most differentially methylated regions after multiple test correction. Using an independent sample of 15 patients and 15 controls from the same population we performed a replication analysis of the most significant CpG sites and pathways. Using an additional case-control sample of 24 individuals from the UK we provided further evidence of association of the DMPs with RA. Finally, in silico datta from a cohort of systemic lupus erythematosus patients (SLE, n=47) and controls (n=56) from the US, we tested the association of the associated DMPs. Results A total of 64 CpG sites were found to be differentially methylated in RA patients compared to controls in the discovery stage after multiple test correction (q Conclusions Our results highlight genes that may drive the pathogenic activity of B cells in RA and suggest shared methylation patterns with SLE. Disclosure of Interest None declared

Epigenetic regulation of immune cell types could be critical for the development and maintenance of autoimmune diseases like rheumatoid arthritis (RA). B cells are highly relevant in RA, since patients express autoantibodies and depleting this cell type is a successful therapeutic approach. Epigenetic variation, such as DNA methylation, may mediate the pathogenic activity of B cells. In this study, we performed an epigenome-wide association study (EWAS) for RA with three different replication cohorts, to identify disease-specific alterations in DNA methylation in B cells. CpG methylation in isolated B lymphocytes was assayed on the Illumina HumanMethylation450 BeadChip in a discovery cohort of RA patients (N = 50) and controls (N = 75). Differential methylation was observed in 64 CpG sites (q

BACKGROUND: Increased secretion of growth hormone leads to gigantism in children and acromegaly in adults; the genetic causes of gigantism and acromegaly are poorly understood. METHODS: We performed clinical and genetic studies of samples obtained from 43 patients with gigantism and then sequenced an implicated gene in samples from 248 patients with acromegaly. RESULTS: We observed microduplication on chromosome Xq26.3 in samples from 13 patients with gigantism; of these samples, 4 were obtained from members of two unrelated kindreds, and 9 were from patients with sporadic cases. All the patients had disease onset during early childhood. Of the patients with gigantism who did not carry an Xq26.3 microduplication, none presented before the age of 5 years. Genomic characterization of the Xq26.3 region suggests that the microduplications are generated during chromosome replication and that they contain four protein-coding genes. Only one of these genes, GPR101, which encodes a G-protein-coupled receptor, was overexpressed in patients' pituitary lesions. We identified a recurrent GPR101 mutation (p.E308D) in 11 of 248 patients with acromegaly, with the mutation found mostly in tumors. When the mutation was transfected into rat GH3 cells, it led to increased release of growth hormone and proliferation of growth hormone-producing cells. CONCLUSIONS: We describe a pediatric disorder (which we have termed X-linked acrogigantism [X-LAG]) that is caused by an Xq26.3 genomic duplication and is characterized by early-onset gigantism resulting from an excess of growth hormone. Duplication of GPR101 probably causes X-LAG. We also found a recurrent mutation in GPR101 in some adults with acromegaly. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others.).

Retinoic acid (RA)-induced expression of bone morphogenetic protein-4 (BMP-4) inhibits in vitro and in vivo cell proliferation and ACTH synthesis in corticotroph-derived tumor cells. Reduced expression of BMP-4 in this adenoma subtype is associated with epigenomic silencing, and similar silencing mechanisms are also associated with the RA-responsive dopamine D2 receptor (D2R) in somatolactotroph cells. We now show that preincubation with the epidrugs zebularine and trichostatin A is obligate and permissive for RA-induced expression of the BMP-4 and the D2R genes in pituitary tumor cells. Combined epidrug challenges are associated with marginal reduction in CpG island methylation. However, significant change to histone tail modifications toward those associated with expression-competent genes is apparent, whereas RA challenge alone or in combined incubations does not have an impact on these modifications. Epidrug-mediated and RA-augmented expression of endogenous BMP-4 increased or decreased cell proliferation and colony-forming efficiency in GH3 and AtT-20 pituitary tumor cells, respectively, recapitulating recent reports of challenges of these cells with exogenous ligand. The specificity of the BMP-4–mediated effects was further supported by knock-down experiments of the BMP-4 antagonist noggin (small interfering RNA [siRNA]). Knock-down of noggin, in the absence and the presence of epidrugs, induced and augmented BMP-4 expression, respectively. In cell proliferation assays, challenge with either epidrugs or siRNA led to significant increase in cell numbers at the 72-hour time point; however, in siRNA-treated cells coincubated with epidrugs, a significant increase was apparent at the 48-hour time point. These studies show the potential of combined drug challenges as a treatment option, where epidrug renders silenced genes responsive to conventional therapeutic options.

Background/Aims: In a genome-wide investigation we recently identified the EGF-containing fibulin-like extracellular matrix protein 1 gene, EFEMP1, as hypermethylated in growth hormone-secreting adenoma. Methods: In an independent cohort we determined expression of EFEMP1, CpG island methylation and histone tail modification status. The causal consequences of epigenetic modification were determined through epidrug-induced reversal and enforced EFEMP1 expression in GH3 cells. Results: The majority of adenomas, irrespective of subtype, show reduced EFEMP1 expression. However, epigenetic change, as determined by CpG island methylation, was not invariantly associated with decreased EFEMP1 expression. Conversely, chromatin immunoprecipitation assays revealed enrichment for modifications associated with either active or silenced genes in adenoma that did or did not express EFEMP1 respectively. In AtT-20 and GH3 cells a causal relationship between epigenetic silencing and expression of EFEMP1 was established where co-incubation with the epidrugs zebularine and TSA induced expression of EFEMP1 and concomitant histone tail modifications toward those associated with expressed genes. Enforced expression of EFEMP1 in GH3 cells was without effect on cell proliferation or apoptotic end-points, however inhibition of endogenous matrix metalloproteinase (MMP)-2 expression was apparent. Primary adenomas did not show this relationship, however a positive correlation was apparent with the MMP7 transcript and perhaps reflects cell or species differences. Conclusions: The protein product of the EFEMP1 gene, fibulin-3, is reported to impact on multiple pathways in a cell-specific context. Subtype-independent loss of EFEMP1 expression in the majority of primary adenomas should prompt more detailed investigation in this tumour type.

Aim: A proof-of-concept study to explore whether DNA methylation at first diagnosis is associated with response to disease-modifying antirheumatic drugs (DMARDs) in patients with early rheumatoid arthritis (RA). Patients & methods: DNA methylation was quantified in T-lymphocytes from 46 treatment-naive patients using HumanMethylation450 BeadChips. Treatment response was determined in 6 months using the European League Against Rheumatism (EULAR) response criteria. Results: Initial filtering identified 21 cytosine-phosphate-guanines (CpGs) that were differentially methylated between responders and nonresponders. After conservative adjustment for multiple testing, six sites remained statistically significant, of which four showed high sensitivity and/or specificity (≥75%) for response to treatment. Moreover, methylation at two sites in combination was the strongest factor associated with response (80.0% sensitivity, 90.9% specificity, AUC 0.85). Conclusion: DNA methylation at diagnosis is associated with disease-modifying antirheumatic drug treatment response in early RA.

The majority of pituitary tumours are monoclonal in origin and arise sporadically or occasionally as part of multiple endocrine neoplasia type 1 (MEN1). Whilst a multi-step aetiology involving both oncogenes and tumour suppressor genes has been proposed for their development, the target(s) of these changes are less clearly defined. Both familial and sporadic pituitary tumours have been shown to harbour allelic deletion on 11q13, which is the location of the recently cloned MEN1 gene. We investigated 23 sporadic pituitary tumours previously shown to harbour allelic deletion on 11q13 with the marker PYGM centromeric and within 50 kb of the MEN1 locus. In addition, the use of intragenic polymorphisms in exon 9 and at D11S4946, and of telomeric loci at D11S4940 and D11S4936, revealed that five of 20 tumours had loss of heterozygosity (LOH) telomeric to the menin gene. However, the overall pattern of loss in informative cases was indicative of non-contiguous deletion that brackets the menin gene. Sequence analysis of all MEN1 coding exons and flanking intronic sequence, in tumours and matched patient leucocyte DNA, did not reveal mutation(s) in any of the 23 tumours studied. A benign polymorphism in exon 9 was encountered at the expected frequency, and in seven patients heterozygous for the polymorphism the tumour showed retention of both copies of the menin gene. Reverse transcription polymerase chain reaction analysis of ten evaluable tumours and four normal pituitaries revealed the presence of the menin transcript. Whilst these findings suggest that gene silencing is unlikely to be mechanistic in sporadic pituitary tumorigenesis, they do not exclude changes in the level or stability of the transcript or translation to mature protein. Our study would support and extend very recent reports of a limited role for mutations in the MEN1 gene in sporadic pituitary tumours. Alternatively, these findings may point to an, as yet, unidentified tumour suppressor gene in this region. © 1999 Cancer Research Campaign

Antiepileptic drugs (AEDs) can lower maternal folate and increase maternal homocysteine levels, which are known to affect the methyl cycle and hence DNA methylation levels. The influence of in utero exposure to AEDs on fetal DNA methylation was investigated. Genome-wide fetal epigenomic profiles were determined using the Infinium 27K BeadArray from Illumina (San Diego, CA, U.S.A.). The Infinium array measures approximately 27,000 CpG loci associated with 14,496 genes at single-nucleotide resolution. Eighteen cord blood samples (nine samples from babies exposed to AEDs and nine controls) from otherwise uncomplicated pregnancies were compared. Unsupervised hierarchic clustering was used to compare the calculated methylation profiles. A clear distinction between the methylation profiles of samples from babies exposed to AEDs in utero compared with controls was detected. These data provide evidence of an epigenetic effect associated with antenatal AED and high-dose folate supplementation during pregnancy. The differences in fetal DNA methylation of those exposed to AEDs shows that a genome-wide effect of methylation is evident. In addition, the epigenetic changes observed appear to be, in this limited sample, independent of extremes of birth weight centiles. These preliminary data highlight possible mechanisms by which AEDs might influence fetal outcomes and the potential of optimizing AED-specific folate supplementation regimens to offset these effects.

Bone morphogenetic protein (BMP)-4 is a key mediator of anterior pituitary organogenesis. However, through inappropriate expression patterns, BMP-4 is also pathogenic in a pituitary adenoma subtype-specific context. In these cases, increase or decrease in BMP-4 in lactotroph- and corticotroph-derived adenomas, respectively, is consistent with a bifunction role for this protein toward either promotion or inhibition of cell proliferation and hormone secretion. To gain insight into the aberrations responsible for differential expression, we examined BMP-4 transcript and protein expression patterns in the major adenomas subtypes. BMP-4 transcript and protein are differentially expressed and show increase in the majority of prolactinomas relative to normal pituitary, whereas the majority of other adenoma subtypes show reduced expression relative to both prolactinoma and normal pituitaries. Reduced expression of BMP-4 is not associated with change in CpG island methylation status. However, histone tail modifications are apparent, as enrichment for a modification associated with silent genes, H3K27me3, and depletion of a modification associated with active genes, H3K9Ac. In pituitary cell lines, reduced BMP-4 expression is also associated with similar histone tail modifications and contemporaneous increase in CpG island methylation. In these cells, coincubation with the demethylating agent zebularine and histone deacetylase inhibitor, trichostatin A, reversed epigenetic changes and restored expression of BMP-4. These studies show that, in contrast to prolactinomas, other adenoma subtypes show reduced expression of BMP-4 where epidrug induced reexpression, alone or in combination with conventional therapies, may offer new treatment strategies.

Global and gene-specific changes in the epigenome are hallmarks of most tumour types, including those of pituitary origin. In contrast to genetic mutations, epigenetic changes (aberrant DNA methylation and histone modifications) are potentially reversible. Drugs that specifically target or inhibit DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) can be used to restore the expression of epigenetically silenced genes. These drugs can potentially increase the sensitivity of tumour cells to conventional treatment modalities, such as chemotherapy and radiotherapy. Drug-induced reversal of transcriptional silencing can also be used to restore dopamine-D(2)-receptor-negative, hormone-refractory tumours to their previous receptor-positive, hormone-responsive status. Synergy between HDAC and DNMT inhibitors makes these pharmacological agents more therapeutically effective when administered in combination than when used alone. Studies in pituitary tumour cell lines show that drug-induced re-expression of the epigenetically silenced dopamine D(2) receptor leads to an increase in apoptosis mediated by a receptor agonist. Collectively, the use of drugs to directly or indirectly reverse gene-specific epigenetic changes, in combination with conventional therapeutic interventions, has potential for the clinical management of multiple tumour types-including those of pituitary origin. Furthermore, these drugs can be used to identify epigenetically regulated genes that could be novel, tumour-specific therapeutic targets.

Epigenetic changes, which target DNA and associated histones, can be described as a pivotal mechanism of interaction between genes and the environment. The field of epigenomics aims to detect and interpret epigenetic modifications at the whole genome level. These approaches have the potential to increase resolution of epigenetic changes to the single base level in multiple disease states or across a population of individuals. Identification and comparison of the epigenomic landscape has challenged our understanding of the regulation of phenotype. Additionally, inclusion of these marks as biomarkers in the early detection or progression monitoring of disease is providing novel avenues for future biomedical research. Cells of the endocrine organs, which include pituitary, thyroid, thymus, pancreas ovary and testes, have been shown to be susceptible to epigenetic alteration, leading to both local and systemic changes often resulting in life-threatening metabolic disease. As with other cell types and populations, endocrine cells are susceptible to tumour development, which in turn may have resulted from aberration of epigenetic control. Techniques including high-throughput sequencing and array-based analysis to investigate these changes have rapidly emerged and are continually evolving. Here, we present a review of these methods and their promise to influence our studies on the epigenome for endocrine research and perhaps to uncover novel therapeutic options in disease states.

Dopamine (DA)-agonist targeting of the DA D2 receptor (D2R) in prolactinomas is the first-line treatment choice for suppression of prolactin and induction of tumor shrinkage. Resistance to DA agonists seems to be related to receptor number. Using the MMQ and GH3 pituitary cell lines, that either do or do not express D2R, respectively, we explored the epigenetic profile associated with the presence or absence of D2R in these cells lines. These studies led us to explore pharmacological strategies designed to restore receptor expression and thereby potentially augment DA agonist-mediated apoptosis. We show in GH3 cells that the D2R harbors increased CpG island-associated methylation and enrichment for histone H3K27me3. Conversely, MMQ cells and normal pituitaries show enrichment for H3K9Ac and barely detectable H3K27me3. Coculture of GH3 cells with the demethylating agent zebularine and the histone deacetylase inhibitor trichostatin A was responsible for a decrease in CpG island methylation and enrichment for the histone H3K9Ac mark. In addition, challenge of GH3 cells with zebularine alone or coculture with both agents led to expression of endogenous D2R in these cells. Induced expression D2R in GH3 cells was associated with a significant increase in apoptosis indices to challenge with either DA or bromocriptine. Specificity of a receptor-mediated response was established in coincubations with specific D2R antagonist and siRNA approaches in GH3 cell and D2R expressing MMQ cell lines. These studies point to the potential efficacy of combined treatment with epigenetic drugs and DA agonists for the medical management of different pituitary tumor subtypes, resistant to conventional therapies.

Folic acid supplementation during pregnancy has known beneficial effects. It reduces risk of neural tube defects and low birth weight. Folate and other one-carbon intermediates might secure these clinical effects via DNA methylation. However, most data on the effects of folate on the epigenome is derived from animal or in vitro models. We examined the relationship between cord blood methylation and maternal folic acid intake, cord blood folate and homocysteine using data from 24 pregnant women. Genome-wide methylation was determined by the level of methylation of LINE-1 repeats using Pyrosequencing. We show that cord plasma homocysteine (p = 0.001, r = -0.688), but not serum folate or maternal folic acid intake, is inverse correlated with LINE-1 methylation. This remained significant after correction for potential confounders (p = 0.004). These data indicate that levels of folate-associated intermediates in cord blood during late pregnancy have significant consequences for the fetal epigenome.

Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to ‘uncover’ or ‘unmask’ silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.

DNA methylation at promoter CpG islands (CGI) is an epigenetic modification associated with inappropriate gene silencing in multiple tumor types. In the absence of a human pituitary tumor cell line, small interfering RNA–mediated knockdown of the maintenance methyltransferase DNA methyltransferase (cytosine 5)-1 (Dnmt1) was used in the murine pituitary adenoma cell line AtT-20. Sustained knockdown induced reexpression of the fully methylated and normally imprinted gene neuronatin (Nnat) in a time-dependent manner. Combined bisulfite restriction analysis (COBRA) revealed that reexpression of Nnat was associated with partial CGI demethylation, which was also observed at the H19 differentially methylated region. Subsequent genome-wide microarray analysis identified 91 genes that were significantly differentially expressed in Dnmt1 knockdown cells (10% false discovery rate). The analysis showed that genes associated with the induction of apoptosis, signal transduction, and developmental processes were significantly overrepresented in this list (P < 0.05). Following validation by reverse transcription-PCR and detection of inappropriate CGI methylation by COBRA, four genes (ICAM1, NNAT, RUNX1, and S100A10) were analyzed in primary human pituitary tumors, each displaying significantly reduced mRNA levels relative to normal pituitary (P < 0.05). For two of these genes, NNAT and S100A10, decreased expression was associated with increased promoter CGI methylation. Induced expression of Nnat in stable transfected AtT-20 cells inhibited cell proliferation. To our knowledge, this is the first report of array-based “epigenetic unmasking” in combination with Dnmt1 knockdown and reveals the potential of this strategy toward identifying genes silenced by epigenetic mechanisms across species boundaries. (Mol Cancer Res 2008;6(10):1567–74)

Each differentiated cell type has its own epigenetic signature, which reflects its genotype, developmental history, and environmental influences, and is ultimately reflected in the phenotype of the cell and organism. Some cells undergo major epigenetic 'reprogramming' during fetal development. The proper, or improper, handling of these highly sensitive periods may have significant short-term and long-term effects on the newborn and his/her progeny. This review highlights the impact of environmental and nutritional factors on the epigenome and the potential effect of epigenetic dysregulation on maternal and fetal pregnancy outcomes, as well as possible long-term implications.

Transgenic mice overexpressing the high mobility group A (HMGA) genes, Hmga1 or Hmga2 develop pituitary tumours and their overexpression is also a frequent finding in human pituitary adenomas. In some cases, increased expression of HMGA2 but not that of HMGA1 is consequent to genetic perturbations. However, recent studies show that down-regulation of microRNA (miRNA), that contemporaneously target the HMGA1 and HMGA2 transcripts, are associated with their overexpression. In a cohort of primary pituitary adenoma we determine the impact of epigenetic modifications on the expression of HMGA-targeting miRNA. For these miRNAs, chromatin immunoprecipitations showed that transcript down-regulation is correlated with histone tail modifications associated with condensed silenced genes. The functional impact of epigenetic modification on miRNA expression was determined in the rodent pituitary cell line, GH3. In these cells, histone tail, miRNA-associated, modifications were similar to those apparent in human adenoma and likely account for their repression. Indeed, challenge of GH3 cells with the epidrugs, zebularine and TSA, led to enrichment of the histone modification, H3K9Ac, associated with active genes, and depletion of the modification, H3K27me3, associated with silent genes and re-expression of HMGA-targeting miRNA. Moreover, epidrugs challenges were also associated with a concomitant decrease in hmga1 transcript and protein levels and concurrent increase in bmp-4 expression. These findings show that the inverse relationship between HMGA expression and targeting miRNA is reversible through epidrug interventions. In addition to showing a mechanistic link between epigenetic modifications and miRNA expression these findings underscore their potential as therapeutic targets in this and other diseases.

Introduction:\ud Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis.\ud \ud Methods:\ud Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases.\ud \ud Results:\ud The ISL1 genes' promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM).\ud \ud Conclusions:\ud In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.

Aims: Alterations in DNA methylation contribute to the abnormal phenotype of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). Herein, we performed genome-wide DNA methylation profiling of RA FLS derived from synovial fluid, a more readily accessible source of disease-associated cells. Patients & Methods: Synovial fluid-derived FLS were isolated and expanded in vitro from 12 patients with RA and osteoarthritis (OA). DNA methylation was interrogated using HumanMethylation450 BeadChips and bisulfite pyrosequencing. Results: Array analysis identified 328 CpGs, representing 195 genes, that were differentially methylated between RA and OA fluid-derived FLS. Comparison with the genes identified in two independent studies of tissue-derived FLS revealed 73 genes in common (~40%), of which 22 shared identity with both studies. Pyrosequencing confirmed altered methylation of these genes. Conclusions: Synovial fluid-derived RA FLS show methylation changes across multiple genes. A subset of these are common with tissue-derived FLS, supporting the use of fluid-derived FLS for future investigations.

In the majority of human cancers, heritable loss of gene function through cell division may be mediated as often by epigenetic as by genetic abnormalities. Epigenetic modification occurs through a process of interrelated changes in CpG island methylation and histone modifications. Candidate gene approaches of cell cycle, growth regulatory and apoptotic genes have shown epigenetic modification associated with loss of cognate proteins in sporadic pituitary tumors. A search for novel genes on the basis of their differential methylation has led to the isolation and functional characterization of a pro-apoptotic mediator--a pituitary tumor apoptosis gene ( PTAG). Although PTAG expression is significantly underexpressed in most pituitary adenomas, mechanisms in addition to methylation most likely account for its loss. The GNAS gene is imprinted in normal pituitary, and activating mutations within Gsalpha, referred to as the gsp oncogene, are almost invariably associated with the maternal expressed allele in somatotrophic adenomas. In addition, epigenetic modification, manifesting as relaxation of imprinting, leads to biallelic expression of Gsalpha irrespective of gsp status. Pituitary tumors as components of familial syndromes represent a rare entity, and the role of epigenetic modification in their evolution and outgrowth is not known. Although speculative, these studies might provide new insight since methylation-associated gene silencing is a feature of other familial tumor types.

Summary background Postsurgical regrowth or recurrence of nonfunctioning pituitary adenomas (NFAs) is not uncommon and often requires further surgery or radiotherapy (DXT). Routine postoperative DXT increases the incidence of hypopituitarism, which is associated with increased morbidity and mortality. Identification of genetic abnormalities in the tumour tissue, which can predict recurrence, may allow targeting DXT to the most appropriate patients. design and methods We have performed loss of heterozygosity (LOH) analysis on 96 NFAs of which 43 (45%) were recurrent and 53 (55%) were nonrecurrent tumours. Analysis of all tumours was performed on the surgical specimen obtained at the time of first surgery. All tumours underwent allelotyping across nine highly informative microsatellite markers selected on the basis of high LOH frequency in an earlier study involving genome-wide allelotyping. LOH frequency across all microsatellite markers as well as across individual markers was compared between the two cohorts of tumours. results LOH frequency in tumours that subsequently recurred was significantly higher across all microsatellite markers as compared to tumours that did not recur (P

Inappropriate expression of cell-cycle regulatory genes and/or their protein products are a frequent finding in pituitary tumours; however, genetic changes associated with or responsible for their dysregulation are in general uncommon. In a search for novel genes, and employing cDNA-representational difference analysis, the gene encoding GADD45gamma was recently isolated and identified as being under-represented in pituitary adenomas. GADD45gamma is a member of a family of genes that are induced by DNA damage and function in the negative regulation of cell growth. In this study, we further confirm this initial report that the majority of pituitary adenomas (22 of 33; 67%) do not express GADD45gamma as determined by RT-PCR analysis. Loss of expression was not associated with either loss of heterozygosity or mutations within the coding region of this gene. In marked contrast, epigenetic change, namely methylation of the GADD45gamma genes CpG island, was a frequent finding (19 of 33 adenoma; 58%) and was significantly associated with tumours in which GADD45gamma transcript was not expressed (18 of 22; 82%; P=0.002). In common with the primary tumours, methylation-associated gene silencing of the GADD45gamma gene was also found in the pituitary tumour cell line AtT20. The treatment of AtT20 cells with the demethylating agent, 5-Aza-2'-deoxycytidine, induced the re-expression of this gene. These findings show that silencing of the GADD45gamma gene in pituitary tumours is primarily associated with methylation of the genes CpG island. Methylation has functional importance since reversal of this epigenetic change in a pituitary-derived cell line is associated with re-expression. Silencing of GADD45gamma, a negative regulator of cell growth, is most likely responsible for conferring a selective growth advantage during tumour evolution and outgrowth.

Throughout the genome CpG dinucleotides are found at one-fifth of their expected frequency and their rarity is further marked by the fact that 70% are methylated. In contrast, CpG islands (CGI), found associated with the promoters of many genes, have maintained their expected frequency of this dinucleotide, and remain unmethylated. Inappropriate methylation of CGIs is associated with histone deacetylation and gene silencing, while methylation of CpGs outside of CGIs is associated with significantly higher mutation rates. Methylation of CGIs is a frequent event in numerous tumour types including those that arise within the pituitary gland. Several studies now show highly frequent methylation of the p16 gene that is significantly associated with loss of cognate protein and that appears to be an early change in pituitary tumorigenesis. Collectively, studies show that somatotrophinomas are an infrequent target for p16 CGI methylation. However, in this pituitary tumour subtype, loss of pRb is associated with either CGI methylation or micro-deletion within the protein-pocket binding domain. As in other tumour types loss of p16 or RB1 appear to be mutually exclusive events in non-functional adenomas and somatotrophinomas respectively. Investigation of the Death Associated Protein Kinase gene shows that loss of its protein (DAPK), a pro-apoptotic molecule, in pituitary tumours is also associated with either methylation or deletion within its associated CGI. In the case of DAPK, however, these changes segregate with invasive pituitary tumours irrespective of tumour subtype. Methylation represents a positive signal that can be detected with exquisite sensitivity; in addition, this change targets multiple genes that show tumour type specificity. Taken together, the detection of DNA methylation changes, using either a panel of predefined marker-islands, or CGI arrays, provides the opportunity to generate "methylation profiles". This new knowledge will increase our understanding of tumour biology and could ultimately aid medical management in these different tumour types, including those of pituitary origin.

Malignant pituitary tumours are rare and their pathogenesis is not fully understood. We have performed genetic analyses on tissues arising from a pituitary carcinoma that initially presented as a silent corticotroph adenoma but which failed to respond to repeated, aggressive surgical and medical therapy. Loss of heterozygosity (LOH) of known or putative tumour suppressor genes (TSG) was assessed by microsatellite analysis of microdissected tumour and matched patient blood DNA. Clonality of the pituitary tumour samples was analysed by two PCR-based techniques; one employing the highly polymorphic short tandem repeat (STR) within the human androgen receptor allele (HUMARA), another based on a restriction fragment length polymorphism of the X chromosome phosphoglycerokinase (PGK-1) gene. Screening with 9 microsatellite markers demonstrated allelic loss at 3 sites (D1S190, D3S1283 and D10S297) in all tumour samples except the presenting pituitary tumour. X chromosome inactivation analysis demonstrated polyclonality in the original presenting tumour and a metastatic deposit but monoclonality in tissue samples from a second and third transsphenoidal resection. In these cases of tumour recurrence both LOH and X chromosome inactivation suggest that monoclonality arose from preferential clonal growth from the original polyclonal tumour. Polyclonality of the metastatic deposit suggests that this was derived from the presenting tumour, although the LOH pattern indicates that a single clone dominates. The data are consistent with increasing allelic loss associated with tumour dedifferentiation and malignant transformation.

Summary In 1992 a 54-year-old man underwent transsphenoidal adenomectomy to remove a clinically nonfunctioning pituitary adenoma during which there was a transient cerebrospinal fluid (CSF) leak. He received radiotherapy to a small residual remnant. Follow-up magnetic resonance imaging (MRI) scan in 1997 showed an increase in the tumour in the pituitary stalk region and an additional intradural lesion at C1 level. In the absence of neurological symptoms and signs, an observational policy was followed. By 1999 the cervical dural lesion had enlarged and laminectomy was performed, during which three intradural lesions were removed. Histology and immunohistochemistry of the metastases were identical to those of the initial pituitary adenoma. Follow-up MRI scan showed extension of the pituitary remnant above the chiasma, requiring transfrontal surgery. Operation was complicated by secondary brain haemorrhage from which the patient died. Autopsy revealed a small amount of residual tumour at the top of the stalk and several small intradural tumour nodules at the level of the foramen magnum. Genetic analysis of the initial pituitary tumour identified significant allelic losses in keeping with its invasive nature, while that of the metastases indicated a separate clone as shown by retention of alleles lost in the primary tumour. The regrown pituitary tumour also appeared to be of a different clone to the initial tumour and the same as two of three of the first metastases (C1 level). The foramen magnum metastasis showed the same loss of heterozygosity (LOH) pattern as one of the original C1 metastases and the pituitary tumour tissue obtained at autopsy. We speculate that at the initial pituitary surgery, cells seeded into the CSF and grew in the dura. These cells were from a different clone, implying that the original pituitary tumour contained at least two clones, possibly three, providing evidence for the contemporaneous oligoclonality of the original pituitary tumour.

Death Associated Protein kinase (DAP kinase) a novel calmodulin-dependent serine/threonine kinase was first identified as a positive mediator of programmed cell death. Loss of DAP kinase expression was first demonstrated in highly metastatic cells, whilst re-expression of the protein resulted in delayed local tumour growth and a decreased incidence of metastasis. Although loss of DAP kinase expression has been reported in several cell lines derived from human malignancies the mechanisms responsible have not been defined. In this study we have examined 32 sporadic pituitary tumours for expression of the DAP kinase protein and transcript. In addition, we examined the methylation and deletion status of the DAP kinase CpG island as possible mechanisms for the inactivation of the DAP kinase gene. Eleven of 32 (34%) tumours had undetectable DAP kinase expression, by Western blot and/or RT-PCR analysis. Loss of DAP kinase expression was significantly (P=0.004) associated with invasive tumours (10 of 17; 59%) compared to their non-invasive (1 of 15; 7%) counterparts. Of 11 tumours that failed to express DAP kinase, five (45%) showed de novo methylation of the CpG island contained within the promoter region, while four (36%) had evidence of homozygous deletion of this region. Statistical analysis showed that loss of DAP kinase expression was significantly (P=

Aims: This study examined links between DNA methylation and birth weight centile (BWC), and explored the impact of genetic variation. Materials & methods: Using HumanMethylation450 arrays, we examined candidate gene-associated CpGs in cord blood from newborns with low (85th centile) BWC (n = 12). Candidates were examined in an investigation cohort (n = 110) using pyrosequencing and genotyping for putative methylation-associated polymorphisms performed using standard PCR. Results: Array analysis identified 314 candidate genes associated with BWC extremes, four of which showed ≥ 4 BWC-linked CpGs. Of these, PM20D1 and MI886 suggested genetically determined methylation levels. However, methylation at three CpGs in FGFR2 remained significantly associated with high BWC (p = 0.004–0.027). Conclusion: We identified a novel biologically plausible candidate (FGFR2) for with BWC that merits further study.

To review recent advances in our knowledge and understanding of aberrations that target the epigenome in sporadic pituitary adenomas.A more complete understanding of the pituitary epigenome has been facilitated by advances in technologies for exploring the tumour-associated epigenomic landscape, and has revealed aberration to the principle targets of these changes, namely, methylation of CpG dinucleotides, modification of histone tails and the expression of target-specific miRNA. Genome-wide investigations, of sporadic pituitary adenoma, have identified novel methylated genes that in some cases are subtype-specific. Recent studies have also shown that silenced genes may be reactivated through epidrug challenges. Moreover, in experimental settings, wherein enforced expression of specific miRNA has been employed, these have been shown to inhibit pituitary cell proliferation in vitro and in vivo.Candidate gene and genome-wide studies reveal frequent epigenetic changes in pituitary adenomas. Aberrations, concurrent with their impact on functional end-points, may display subtype specificity, whereas others appear to be independent of adenoma subtype. Changes to the epigenomic landscape, and apparent as CpG island methylation and/or as histone tail modifications, show sensitivity to epidrug-induced re-expression that concomitantly impacts on cell proliferation. Similarly, enforced expression of silenced miRNA in model systems is also associated with similar end-points. Collectively, emerging data show that these types of manipulation, alone or in combination with a more conventional therapeutic option, offer new avenues for the medical management of these tumours.

Email: w.e.lalien@keele.ac.uk The cyclin D1 (CCND1) gene contains a frequent A/G polymorphism within the splice donor region of exon 4/intron 4. CCNDI genotype is associated with clinical outcome in a number of malignancies although prognostic significance varies with tumour type. We examined CCND1 allele frequencies and genotype distribution in 294 patients with sporadic pituitary adenomas of various histologies. CCNDI allele frequencies and distribution of genotypes were similar in the 294 cases compared with previously reported control populations. Analysis according to tumour subtype showed no statistical difference in allele frequencies compared with controls. However, CCNDI genotype distribution in the somatotrophinomas showed a significant difference compared with normal controls (P = 0.008). We next examined CCNDI allele frequencies and genotype distribution across the tumour grades. Within the total tumour cohort the CCNDI allele frequencies showed a significant inverse relationship across the tumour grades (P = 0.005). The CCNDI A allele progressively increased from grade 1 (0.37) through to grade 4 (0.62) tumours, whilst the CCNDI G allele frequency progressively decreased from grade 1 (0.63) through to grade 4 (0.38) tumours. Trend analysis of CCND1 genotypes showed a significant progressive increase in AA frequency from grade 1 (15%) through to grade 4 (46%) tumours (P = 0.005). The CCNDI GG genotype progressively decreased from grade 1 (41%) through to grade 4 (23%) tumours (P = 0.204). No statistical significance was observed between CCNDI AG genotype and tumour grades. While the functional significance of the observed segregation of the CCNDI A/G polymorphism and tumour grade is unclear, our data suggest that CCNDI allele frequencies and genotype distributions show significant differences between tumour grades in sporadic pituitary adenomas. Since CCNDI genotype may be determined by analysis of peripheral blood samples it may provide a useful predictive marker for those tumours likely to show invasive behaviour. This may be clinically useful in indicating which tumours should receive adjunctive treatment (e.g. radiotherapy) immediately after surgical resection.

The pathogenetic mechanisms underlying pituitary tumorigenesis are largely unknown. Previous reports have suggested that aggressive pituitary adenomas and/or carcinomas may be associated with genetic alterations that are distinct from those responsible for the more common and less aggressive pituitary adenomas. Here, we describe the clonal composition of a pituitary carcinoma, its recurrence and its metastasis. The samples studied were from a 48-year-old woman who presented with recurrent Cushing's syndrome. During the 8-year course of her disease, she had an ACTH-producing pituitary carcinoma requiring two transsphenoidal procedures and resection of a metastatic cervical lymph node. Her disease remained active despite surgical resection, external beam irradiation and medical treatment with ketoconazole. Ultimately, bilateral adrenalectomy was performed to control the hypercortisolism. Morphological and immunohistochemical studies revealed that the primary and recurrent pituitary tumours and the metastatic lesion were an endocrine tumour with ACTH and growth hormone immunoreactivity. Primary, recurrent and metastatic tumour DNAs were analysed for X-chromosome inactivation and loss of heterozygosity (LOH) at several microsatellite loci on chromosomes 9,10, 11, 13 and 22. All three lesions were monoclonal in composition as suggested by the pattern of X chromosome inactivation of the PGK-1 allele. Moreover, the primary, recurrent and metastatic lesions demonstrated LOH at the microsatellite allelic markers PYGM and D10S217. In contrast, however, the metastatic lesion showed a loss-to-retention pattern at two distinct loci (IFNA and D22S156) compared to the primary and recurrent pituitary tumours. These findings, while consistent with a clonal composition of the primary and metastatic pituitary lesions, show each clone to be distinct. This is the first description of a metastatic pituitary carcinoma with a distinct clonal composition from its primary source.

Components of the pRb/p16/cyclin D1/CDK4 pathway are frequent targets in numerous tumour types, including those of pituitary origin. However, previous studies of pituitary tumours have examined individual components of this pathway. Therefore, to determine their overall contribution we have simultaneously examined the immunohistochemical status of pRb, p16 and cyclin D1 and analysed the CDK4 gene for a characterized activating mutation. Of the total pituitary tumour cohort (29 clinically non-functioning adenomas and 16 somatotrophinomas) abnormal expression of either pRb, p16 or cyclin D1 was observed in 36 of 45 (80%) tumours and was significantly (P = 0.005) associated with non-functioning tumours (27/29; 93%) compared with somatotrophinomas (9/16, 56%). Loss of either pRb or p16 expression was mutually exclusive in 23 of 45 (51%) tumours, whilst concomitant loss of pRb and p16 expression was observed in five tumours. Cyclin D1 overexpression was observed in 22 of 45 (49%) tumours, however, there was no significant association between overexpression of cyclin D1 and the expression status of either pRb or p16. In addition, no activating mutations within codon 24 of the CDK4 gene were detected. This study provides evidence for the first time that components of the pRb/p16/cyclin D1/CDK4 pathway, either alone or in combination, are frequently deregulated in human pituitary tumours, suggesting that this pathway may be a useful target in drug or gene therapeutic approaches.

Pituitary tumours are a common type of intracranial neoplasm and, depending on the cell type of origin, have diverse endocrine and reproductive effects. The developmental biology of the different cell types is understood to result from a sequential activation of a cascade of transcription factors, and mutations in these factors result in various forms of hypopituitarism. Tumours in the pituitary gland arise from activation of dominantly acting oncogenes such as gsp, or from loss of function of a series of tumour suppressor genes such as MEN1. Abnormal patterns of DNA methylation may be implicated in the allelic losses that cause tumour suppressor gene silencing. The different clinically recognized types of pituitary tumour are currently treated by medical therapies such as dopamine and somatostatin agonists, surgery or radiotherapy. However, these treatments are not entirely satisfactory and recent advances in gene therapy may offer valuable new therapeutic opportunities for patients with aggressive tumours that fail to respond to traditional approaches.

Introduction Colorectal cancer is the commonest cause of death due to malignancy in non-smokers in the western countries. The two main hereditary types of colorectal cancer are familial adenomatous polyposis (FAP) and hereditary non-polyposis colorectal cancer (HNPCC), constituting approximately 10% of all cases of colorectal cancer. The main aim of this review is to reappraise the current advances in the genetics and diagnosis of HNPCC. Methods A Medline search was carried out to identify papers published from 1970 to 1999 on HNPCC. Embase and Cochrane databases were also searched. Reference lists of retrieved articles were carefully searched for additional articles. Results and conclusions Recent technological advances in the genetics of HNPCC have refined the criteria for diagnosis and management of HNPCC, however current policies regarding the testing of pedigrees are not clearly established. We believe that with the rapid development in this area definitive clinical guidelines will need to be available in future for the management of HNPCC.

Pituitary tumors are the result of a monoclonal outgrowth where the intrinsic genetic defects involve oncogenes, tumor suppressor genes (TSG), and most likely genes responsible for differentiation. In addition, hypothalamic and intrapituitary derived growth factors are imposed upon these aberrant cells, contributing to their growth characteristics. While histological examination will not identify those tumors likely to progress toward an invasive phenotype or those destined toward recurrence recent advances in the molecular pathology of these tumors holds significant promise for prediction of recurrence and the design of novel treatment strategies. Moreover, emerging data clearly indicate that different molecular mechanisms are involved in the pathogenesis of the various pituitary tumor subtypes. Until recently the gsp oncogene was the only oncogene significantly associated with pituitary tumors; however, emerging data have describe a role for PTTG and cyclin D1 in pituitary tumorigenesis. For known and putative TSG loci, allelic losses on the long arms of chromosomes 10, 11, and 13 are significantly associated with the transition from the noninvasive to the invasive and metastatic phenotype, while losses on chromosome 9p occur early in pituitary tumorigenesis. Studies of known TSG at these loci, including the menin gene and RB1, would suggest a limited role, if any, in pituitary tumors. However, loss of pRB is evident in a proportion of somatotropinomas but is not associated with allelic loss of an RB1 intragenic marker. The gene encoding p16/CDKN2A is neither deleted nor mutated in pituitary tumors; however, its associated CpG island is frequently methylated and is associated with a loss of p16 protein expression. Allelic losses on chromosome 9p, frequent methylation, and loss of p16 protein appear as early changes in nonfunctional tumors, whereas they are infrequent events in somatotropinomas. The functional consequence of enforced expression of p16/CDKN2A in the mouse corticotroph cell line AtT20 has shown that it is responsible for a profound reduction in cell proliferation and the mechanism is a G(1) arrest, mimicking the in vivo role of this cell cycle regulator in most tissues. The combined data from several groups show that the allelic losses reported at known TSG loci are not accompanied by mutation in the retained allele. However, since abnormal methylation patterns may precede and predispose toward genetic instability this could account for the allelic losses on these chromosomes. Equally, since DNA methylation may lead to reduced expression of a gene it might also account for the reduced expression of as yet unidentified TSGs implicated in pituitary tumorigenesis. Collectively these studies hold significant promise as markers predictive of tumor behavior and point to novel treatment strategies, which may include the reactivation of TSGs that are intact but silenced through epigenetic mechanisms.

During batch growth of two recombinant NS0 myelomas, an increase in the expression of the endoplasmic reticulum (ER) proteins (GRP78/BiP, GRP94, and ERp72) was observed. A marked increase in these proteins was associated with the decline phase of growth, an increase in the production rate of chimeric antibody, and a marked slowing or halt in the uptake of glucose and glutamate. Refeeding with glucose, glutamate, or a mixture of amino acids just prior to the onset of decline phase failed to repress induction. Although refeeding with glutamate led to an increase in specific productivity, there was no significant difference in the pattern of ER protein induction. These results indicate that an increase in ER protein expression is not solely related to productivity but also to certain changes that occur during the course of batch growth.

Methylation is essential for embryonic development, however aberrant methylation of CpG islands associated with the tumour suppressor genes (TSGs) and leading to gene silencing is found in numerous tumour types. The TSG p16/CDKN2A is involved in the genesis of many tumour types and frequent methylation of the CpG island of the p16/CDKN2A gene is associated with loss of protein expression in pituitary tumours. In addition, CpG sites are mutational hotspots and abnormal methylation patterns have been shown to lead to genetic instability, predisposing to, and preceding allelic loss. Although several studies of pituitary tumours have shown loss of genetic material at known and putative TSGs loci, studies of the retained alleles have revealed infrequent mutation. Equally, for several other TSGs no mechanisms have been described for their reduced expression. Methylation may represent a unifying theme, responsible in some cases for an absence or reduced expression and in other cases predisposing to allelic loss that may or may not encompass a TSG. In several tumour types treatment of tumours or their cognate cell lines with demethylating agents induces expression of previously methylated genes. Using the mouse corticotroph cell line AtT20 as a model system, transfection studies showed restoration of growth control through induction of ectopically expressed p16/CDKN2A. These effects were reversed by prior in vitro methylation of the constructs' CpG sites within the coding region of this gene. Methylation of an otherwise unmethylated CpG island renders a gene transcriptionally incompetent and clinically these genes represent attractive therapeutic targets since the gene is neither lost nor mutated, but may be reactivated. Future studies will no doubt describe more efficacious pharmacological interventions and identify the mechanisms responsible for the abnormal methylation patterns seen in tumours including those of pituitary origin.

Corticotropinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance. As cortisol resistance can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotropinomas to cortisol is also caused by de novo mutations in the GR gene. We screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing’s disease for mutations using PCR/single strand conformation polymorphism analysis. In a previous study, we identified 5 polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus. Except for 1 silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotropinomas from these patients. Of the 22 patients, 18 were heterozygous for at least 1 of the polymorphisms. In 6 of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only 1, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically nonfunctioning, 8 prolactinomas, and 9 GH-producing adenomas) and found evidence for LOH in only 1 prolactinoma. In six patients LOH was found at the GR gene locus (chromosome 5) in DNA derived from adenoma cells. Our observations indicate for the first time that LOH at the GR gene locus is a relatively frequent phenomenon in pituitary adenomas of patients with Cushing’s disease. This might explain the relative resistance of the adenoma cells to the inhibitory feedback action of cortisol on ACTH secretion. The specificity of the GR LOH to corticotropinomas supports this concept. Somatic mutations of the GR are not a frequent cause of relative cortisol resistance in these cells.