Biomedical subjects

Norman L Eberhardt

Publications and source records attributed to Norman L Eberhardt.

16 recordsLinked to original sources

PubMed2006

hMLH1 promoter methylation and silencing in primary endometrial cancers are associated with specific alterations in MBDs occupancy and histone modifications.

OBJECTIVE: To investigate the relationship between hMLH1 promoter methylation and changes in chromatin composition. To study how the occupancy of methyl CpG binding domain proteins (MBDs) and histone acetylation/methylation in hMLH1 promoter may participate in hMLH1 silencing. METHODS: 64 endometrial cancer samples were screened for hMLH1 mRNA expression. hMLH1 promoter methylation status was confirmed by methylation-specific PCR in cancers with high and low levels of hMLH1 expression. Chromatin immunoprecipitation was performed to compare the MBD occupancy and histone modifications between the methylated/silenced and unmethylated/active hMLH1 genes in multiple primary endometrial cancers. RESULTS: We demonstrated that MeCP2, MBD1 and MBD2, but not MBD3 and MBD4, specifically bind to methylated hMLH1 promoters. Hyperacetylated histones H3 and H4 were found to be associated with the unmethylated and transcriptionally active hMLH1 promoters. While H3 lysine-4 methylation was present in unmethylated hMLH1 promoters, H3 lysine-9 methylation was found exclusively in methylated promoters. Western blot analysis showed that similar global levels of MBDs and histones were present in the two cancer groups with high and low hMLH1 expression. CONCLUSIONS: A distinct combination of MBDs and histone modification is associated with the silencing of the hMLH1 gene. The changes in hMLH1 chromatin composition are closely related to methylation status of hMLH1 promoters. These changes are not accounted by the global expression levels of MBDs and histones in endometrial cancers.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Histone deacetylase inhibitors decrease DNA methyltransferase-3B messenger RNA stability and down-regulate de novo DNA methyltransferase activity in human endometrial cells.

It is well known that the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) acts synergistically with the DNA methyltransferase (DNMT) inhibitor 5-aza-2'-deoxycytidine (ADC) to reactivate DNA methylation-silenced genes. Moreover, in several studies, TSA was capable of inducing DNA demethylation even in the absence of ADC. Here we describe a mechanism by which HDAC inhibitors affect DNA methylation through their regulation on DNMT3B, a methyltransferase responsible for de novo DNA methylation. Using quantitative real-time PCR and Western blot analysis, we show that TSA down-regulates DNMT3B mRNA and protein expression in human endometrial cancer cells. This decrease in DNMT3B mRNA results in a significant reduction in de novo methylation activities. Further experiments indicated that TSA decreases DNMT3B mRNA stability and reduces its half-life from approximately 4 to 2.5 hours. We established that protein synthesis is required for posttranscriptional regulation, suggesting the involvement of an RNase and/or key mRNA stabilization factor(s) controlling the DNMT3B mRNA stability. Therefore, TSA may not only modify histone acetylation, but also potentially alter DNA methylation. Since the HDAC inhibitors are frequently used in epigenetic studies and are considered to be promising anticancer drugs, these new findings will have implications in both laboratory and clinical settings.

Azacitidine↗

PubMed2005

Up-regulation of DNA methyltransferase 3B expression in endometrial cancers.

OBJECTIVE: To understand the role of epigenetic regulation in the pathogenesis of endometrial cancer, we have characterized DNA methyltransferase 3B (DNMT3B) gene expression in normal, Grade I and Grade III endometrioid cancers, and examined DNMT3B promoter activities in endometrial cancer cell lines. METHODS: DNMT3B expression was measured in normal, Grade I, and Grade III endometrioid cancer samples. Real-time PCR and Western blot analysis were performed to compare DNMT3B mRNA and protein levels. DNMT3B levels were also compared among endometrial cell lines including those for Ishikawa, KLE, AN3, RL-95, HEC-1A, and HEC-1B. DNMT3B promoter reporter plasmids were constructed. Promoter activities in well and poorly differentiated cell lines were compared by in vitro reporter gene transfection. RESULTS: DNMT3B was significantly up-regulated in both Grade I and Grade III cancers as compared to normal controls. Western blot analysis confirmed the increased DNMT3B protein expression in cancer tissues. It was also found that the well-differentiated endometrial cell line, Ishikawa, expressed lower levels of DNMT3B than the poorly differentiated KLE cells, the expression patterns similar to those observed in tumor specimens. CONCLUSION: The results suggest that DNMT3B overexpression may play a significant role in endometrial cancer development. In addition, the transfection experiments indicated that DNMT3B promoters are more active in the poorly differentiated endometrial cancer cell lines, suggesting that the in vitro assay provides a useful model for studying the DNMT3B transactivation mechanism related to tumor transformation.

Carcinoma, Endometrioid↗

PubMed2005

Opposite alterations of DNA methyltransferase gene expression in endometrioid and serous endometrial cancers.

OBJECTIVE: To examine the DNA methyltransferase (DNMT) mRNA and protein levels in endometrioid and serous cancers and to study the relationship between DNA methyltransferase expression and endometrial cancer development. METHODS: Normal endometrium, Grade I and Grade III endometrioid carcinoma tissues and cell lines, as well as serous cancer tissues, were analyzed for DNMT expression. Real-time PCR and Western blot techniques were employed to measure the mRNA and protein levels of the four DNA methyltransferases, DNMT1, DNMT2, DNMT3A, and DNMT3B. Immunohistochemistry was performed to detect alterations in DNMT nuclear localization and spatial organization patterns. RESULTS: While DNMT2 and DNMT3A expression appear to be normal, two- to fourfold increase in DNMT1 and DNMT3B were found in both Grade I and Grade III endometrioid cancers. In addition, the poorly differentiated cell lines expressed relatively higher DNMT levels than well-differentiated cells. In contrast to endometrioid carcinomas, serous cancers expressed substantially lower levels of DNMT1 and DNMT3B than normal controls, with four- and twofold reduction observed in DNMT1 and DNMT3B mRNA levels, respectively. Western blot analysis confirmed opposite expression patterns of DNMT1 and DNMT3B protein in endometrioid and serous cancers. Immunohistochemistry showed normal nuclear localization of DNMT1 and DNMT3B in Type I and Type II cancer specimens as well as cell cultures. CONCLUSION: Two opposite DNMT expression patterns were identified in endometrioid and serous cancers. The concerted upregulation in maintenance and de novo DNA methyltransferases in endometrioid carcinomas is consistent with a tendency for gene-specific hypermethylation observed in this histologic subtype, and may be implicated in tumor suppressor silencing. In contrast, the downregulation of maintenance and de novo DNA methyltransferases in serous cancers suggests that these tumors may contain hypomethylated genomic DNA, which has been associated with a higher mutation rate and is consistent with the known pathogenesis of serous-specific phenotypes. Taken together, the data suggest that divergent DNA methylation pathways may be implicated in the development of Type I and Type II endometrial cancers.

Carcinoma, Endometrioid↗

PubMed2005

Epigenetic-mediated upregulation of progesterone receptor B gene in endometrial cancer cell lines.

OBJECTIVES: To determine if epigenetic interference can restore progesterone receptor-B (PR-B) expression in PR-B negative endometrial adenocarcinoma cell lines, and to characterize the kinetics of PR-B induction mediated by DNA methyltransferase and histone deacetylase inhibitors. METHODS: The PR-B negative endometrioid cancer cell lines KLE and HEC-1B were used as study models. PR-B mRNA and protein expression levels were measured using real-time PCR and Western blot analysis, respectively. DNA methylation levels of the PR-B promoter were determined by methylation-specific PCR. Dose-response correlations and the duration of response to aza-deoxycytidine (ADC) and trichostatin A (TSA) were characterized. Cell responses to prolonged and repeated drug treatment were also examined. RESULTS: Relatively low concentrations of ADC and TSA over a 24-h period induced PR-B expression. Furthermore, ADC and TSA acted synergistically to reactivate PR-B expression. Depending on the cell line used, PR-B mRNA was induced 10-110 fold. This elevated PR-B expression continued for 48 h after drug withdrawal. Sustained upregulation of PR-B mRNA and protein was observed during prolonged and repeated drug treatment. CONCLUSION: The epigenetically silenced PR-B gene remains sensitive to changes in DNA demethylation and histone acetylation in uterine adenocarcinoma cell lines. Treatment with ADC and/or TSA results in a robust and sustainable PR-B upregulation. These small molecule epigenetic modifying agents may be used to sensitize poorly differentiated, PR-B negative endometrial cancers to progestational therapy.

Adenocarcinoma↗

PubMed2004

PPARgamma staining as a surrogate for PAX8/PPARgamma fusion oncogene expression in follicular neoplasms: clinicopathological correlation and histopathological diagnostic value.

The PAX8/PPARgamma (PPFP) fusion-oncogene is moderately specific for follicular thyroid carcinomas (FTC). It remains unknown whether this can be translated into improved diagnosis, classification, or outcome prediction. We studied a cohort of well-characterized follicular adenomas (FA), FTC, and Hurthle cell carcinomas (HCC) from patients with complete clinical follow-up, to determine whether PPARgamma immunohistochemistry (as a surrogate of PAX8/PPARgamma expression) helps to distinguish FA from FTC and to assess its diagnostic accuracy as an adjunct to frozen section. We also correlated PPARgamma staining with clinical outcomes to assess its role as a prognostic marker.PPARgamma staining was more common in FTC (31 of 54; 57%) than in HCC (one of 23; 4%) or FA (four of 31; 13%) (P < 0.000001). Adjunctive use of PPARgamma immunohistochemistry improved diagnostic sensitivity of intraoperative frozen section from 84% to 96% (P < 0.05) but reduced specificity from 100% to 90% (P < 0.05). PPARgamma staining was associated with favorable prognostic indicators (female gender, better tumor differentiation, and lesser risk of metastases).PPARgamma staining may be helpful in the differential diagnosis of FA, FTC, and HCC, particularly when diagnostic sensitivity of histomorphology is reduced (e.g. during intraoperative frozen section). PPARgamma staining also shows an association with favorable prognosis and may have a role in risk stratification.

Adenoma↗

PubMed2004

The PAX8/PPARgamma fusion oncoprotein transforms immortalized human thyrocytes through a mechanism probably involving wild-type PPARgamma inhibition.

Follicular thyroid carcinoma (FTC) frequently harbors the PAX8/PPARgamma fusion gene (PPFP); however, its oncogenic role and mechanism(s) of action remain undefined. We investigated PPFP's effects on cell growth, apoptosis, cell-cell, and cell-matrix interactions in immortalized human thyroid cells (Nthy-ori 3-1) and NIH 3T3 cells. PPFP expression increased the growth of transient and stable Nthy-ori transfectants ( approximately threefold by 72 h). There was an 8.4% increase of cells in the S+G2/M phase, a 7.8% decrease in cells in the G0+G1 phase and a 66% decline in apoptosis at 72 h. Stable Nthy-ori PPFP transfectants grew in soft agar, and PPFP-transfected NIH 3T3 cells exhibited efficient focus formation, suggesting loss of anchorage-dependent growth and contact inhibition, respectively. Overexpression of PPARgamma in Nthy-ori cells did not recapitulate PPFP's growth effects. Treatment of Nthy-ori cells with an irreversible PPARgamma inhibitor mimicked the growth-promoting effects of PPFP and co-expression of PPFP and PPARgamma blocked PPARgamma transactivation activity. Our data provide functional evidence that PPFP acts as an oncoprotein, whose transforming properties depend in part on inhibition of PPARgamma. Our data suggest that PPFP contributes to malignant transformation during FTC oncogenesis by acting on several cellular pathways, at least some of which are normally regulated by PPARgamma.

Apoptosis↗

PubMed2004

The PAX8/PPAR gamma fusion oncogene as a potential therapeutic target in follicular thyroid carcinoma.

Follicular thyroid carcinoma (FTC) accounts for approximately 20% of all thyroid cancers, and up to 40% of the deaths associated with this disease. Current treatment approaches include surgery, followed by radioactive iodine therapy. However, a significant proportion of locally advanced and metastatic FTC fails to concentrate iodine. Because traditional chemotherapeutic agents have not been shown to alter outcomes in this disease, novel therapeutic strategies are needed for advanced disease. Recently, a genomic rearrangement has been identified in up to 50% of FTC, involving a translocation event between chromosome regions 3p25 and 2q13. This translocation fuses the thyroid-specific transcription factor PAX8 gene with the PPARgamma gene, a ubiquitously expressed transcription factor. We have confirmed that this Pax8/PPARgamma fusion gene (designated PPFP) is an oncogene, which accelerates cell growth, reduces rates of apoptosis and permits anchorage independent and contact uninhibited growth of a thyroid cell line. The action of PPFP arises, at least in part, through its activity as a dominant-negative inhibitor of the wild-type PPARgamma transcription factor. Although the mechanism by which PPFP impairs PPARgamma activity remains unknown at this time, it is likely to be mediated by competition for the genomic PPARgamma response elements, the endogenous ligand, or various cofactors, including the Retinoid X Receptor (RXR). Consequently, modulation of PPFP activity might be possible through the use of PPARgamma agonists, RXR-agonists, or specific modulators of PPFP itself. Alternatively, modulation of several down-stream regulatory pathways may become possible, as the consequences of PPARgamma inhibition become better known. PPFP represents a potential novel target for the management of advanced FTC.

Adenocarcinoma, Follicular↗

PubMed2003

A novel dynamin III isoform is up-regulated in the central nervous system in hypothyroidism.

Hypothyroidism in early postnatal development leads to abnormal CNS development that may be controlled in part at the level of gene transcription. Comparing the expression of euthyroid (EuT) and hypothyroid (HypoT) rat brain mRNAs by differential display PCR (ddPCR), we identified a novel dynamin III mRNA that was up-regulated in the hypothyroid state. Northern analysis of brain mRNA using a probe from the dynamin III open reading frame (ORF) revealed two transcripts of 3.0 and 7.2kb size. The 3.0 kb transcript was observed in testis and brain, but not liver or lung RNA. In the brain the 3.0 kb transcript increased from 25 to 57% of adult (Ad) levels from postnatal day (p) p2-p15, but was not significantly regulated by thyroid hormone status. In contrast, the more abundant 7.2 kb transcript increased from 16.8 to 48.0% of adult levels from p2 to p15 in euthyroid rat pups but from 54.0% of adult levels at p2 to 97.9% of adult levels by p15 in hypothyroid pups. Overlapping cDNA clones from a rat brain cDNA library defined the 7.2kb mRNA, which consisted of the complete ORF, containing a four amino acid insert at the end of the pleckstrin homology domain (PHD), and two unique 3'-flanking regions, that are likely derived from alternative processing. Thus, the 7.2 kb dynamin III transcript is brain-specific and selectively regulated by thyroid hormone status. The data suggest that the regulation of dynamin III by altered thyroid hormone status may affect synaptogenesis in the CNS through dynamin's essential roles in synaptic vesicle and receptor recycling, neurotransmitter reuptake, and growth factor receptor signaling.

Amino Acid Sequence↗

PubMed2003

Clustering of the B cell receptor is not required for the apoptotic response.

We examined the role of BCR cell membrane redistribution in anti-IgM-induced apoptosis in three human B cell lines, RA#1, 2G6, and MC116, that differ in their relative levels of sIgM expression. The apoptotic response was found to be dependent on the nature of the anti-IgM and the cell line. In the cell lines, RA#1 and MC116, sIgM aggregated into patches that were insensitive to the disruption of cholesterol-rich membrane microdomains by nystatin or beta-MCD. The B cell line 2G6 was able to reorganize sIgM into a tight coalescent cap upon anti-IgM treatment. However, in this case, the lipid raft inhibitors nystatin and beta-MCD disrupted the patching. In 2G6 cells, BCR-mediated apoptosis was not affected by nystatin treatment, whereas it increased in beta-MCD pretreated cells. Thus, no evident correlation was found between apoptosis and BCR cell membrane redistribution or lipid raft formation in either of the three cell lines. The data indicate that the apoptotic signal transduction pathway is independent of BCR translocation into lipid rafts and/or aggregation.

Antibodies, Anti-Idiotypic↗

PubMed2003

Kinetics of the apoptotic response induced by anti-IgM engagement of the B cell receptor is dependent on the density of cell surface immunoglobulin M expression.

The effect of B cell receptor (BCR) density on anti-BCR-induced apoptosis was assessed in Ramos cell lines, expressing low, medium, or high levels of surface IgM (sIgM(LO), sIgM(MED), sIgM(HI)). All cells required a 6-mug/ml threshold of anti-IgM to elicit apoptosis. Anti-IgM treatment of sIgM(LO) cells induced growth inhibition and limited dose-independent apoptosis. Anti-IgM treatment of sIgM(MED) cells induced dose-independent death with a 32-h lag. Ligation of the BCR in the sIgM(HI) cells induced rapid apoptosis beginning by 6 h, which was dose-dependent. Secondary crosslinking reagents did not affect apoptosis, and this effect was independent of anti-IgM concentration, time, or sIgM density. These results suggest that the response to BCR engagement strongly depends on the cell surface receptor density.

Antibodies, Anti-Idiotypic↗

PubMed2003

Thyroid gland clonality revisited: the embryonal patch size of the normal human thyroid gland is very large, suggesting X-chromosome inactivation tumor clonality studies of thyroid tumors have to be interpreted with caution.

It is widely assumed thyroid carcinomas, adenomas, and many hyperplastic nodules are monoclonal. This belief is based on X-chromosome inactivation analyses of thyroid tumors. However, X-chromosome inactivation studies of tumors are informative only when interpreted in the context of the clonal composition of the surrounding normal tissue, and in the case of thyroid tissue, such analyses have never been systematically performed in humans. The aim of this study was to determine the embryonal patch size of the human thyroid gland. We performed human androgen receptor (HUMARA) assay-based X-chromosome inactivation analysis on 20 microdissected normal thyroid specimens from 16 female subjects. Monoclonality was observed in 70% of tested specimens, and polyclonal X-inactivation patterns were present in only 30% of specimens. According to our results the monoclonal patch size of normal human thyroid tissue is between 48 mm(2) and 128 mm(2) (1-4 x 10(5) thyrocytes). Our data indicate that normal thyroid epithelium is organized into large stem cell-derived monoclonal patches. Therefore, monoclonality in neoplastic and hyperplastic lesions may just be a reflection of normal thyroid epithelium clonal composition.

Adult↗

PubMed2003

Optimising restriction enzyme cleavage of DNA derived from archival histopathological samples: an improved HUMARA assay.

AIMS: Our aim was to evaluate and optimise methylation-sensitive restriction enzyme assays for use in formalin-fixed, paraffin-embedded tissue (FFPET) samples, in order to improve the application of the HUMARA X-chromosome inactivation assay to FFPET samples. METHODS: We extracted DNA from normal male colon and thyroid FFPET. Several DNA clean-up procedures and restriction enzyme buffer compositions were tested for two methylation-sensitive enzymes, HhaI and HpaII and a non-methylation-sensitive isoschizomere, MspI. RESULTS: By including both a non-methylation-sensitive control enzyme and DNA from male archival specimens in our experiments, we were able to detect even subtle degrees of incomplete digestion. We showed that FFPET-derived DNA is a poor substrate for restriction enzymes, especially for methylation-sensitive restriction endonucleases. An optimised DNA clean-up protocol and restriction enzyme buffer-mix allowed us to achieve complete digestion. CONCLUSIONS: The combination of multiple, rigorous controls, DNA clean-up and restriction buffer optimisation increases the reliability of HUMARA-based X-chromosome inactivation analysis of FFPET samples. Analogous approaches are likely to allow optimisation of other restriction enzyme-based assays of FFPET samples.

Colon↗

PubMed2002

Seeing through the face of deception.

We have developed a high-definition thermal-imaging technique that can detect attempted deceit by recording the thermal patterns from people's faces. This technique has an accuracy comparable to that of polygraph examination by experts and has potential for application in remote and rapid security screening, without the need for skilled staff or physical contact.

Body Temperature↗

PubMed2002

Loss of heterozygosity studies revisited: prior quantification of the amplifiable DNA content of archival samples improves efficiency and reliability.

Polymerase chain reaction (PCR)-based loss of heterozygosity (LOH) studies of archival formalin-fixed, paraffin-embedded (FFPE) tumor tissues have become an important tool in the search for tumor suppressor genes and oncogenes and are also used increasingly in clinical practice. However, FFPE tissue samples may contain little amplifiable DNA, resulting in frequent reaction failures and unreliable LOH data. Using pairs of serial dilutions of reference DNA, we determined the minimum amplifiable DNA concentration necessary for reliable microsatellite-PCR LOH analysis. We then measured the amplifiable DNA content of a selection of frozen and FFPE-derived tumor specimens by real-time quantitative PCR. A minimum input of 600 pg of 100% amplifiable DNA per PCR was required for reliable LOH analysis. While the total DNA concentrations of all samples exceeded this figure, most FFPE-sample-derived DNA was non-amplifiable, with ratios of actually amplifiable DNA to total DNA as low as 1 to 3625. Many FFPE samples therefore contained substantially less than 600 pg/microl of actually amplifiable DNA, making them potentially unsuitable for LOH studies. Real-time quantitative PCR before LOH studies of FFPE tissues allows: identification of samples, which will fail microsatellite-PCR; exclusion of samples, which will yield unreliable results; and optimal adjustment of template input for the remainder. Amplification reactions undertaken without this precaution can result in unreliable LOH data.

DNA↗

PubMed2002

High resolution loss of heterozygosity mapping of 17p13 in thyroid cancer: Hurthle cell carcinomas exhibit a small 411-kilobase common region of allelic imbalance, probably containing a novel tumor suppressor gene.

There is strong evidence in many tumor types, including thyroid cancer, for a novel tumor suppressor gene (TSG) at 17p13. To identify the putative thyroid 17p13 TSG we mapped thyroid tumor loss of heterozygosity (LOH) at high resolution within this region. We examined 20 typical follicular thyroid carcinomas (FTC), 19 Hurthle cell carcinomas (HCC), 15 papillary thyroid carcinomas (PTC), and 7 follicular adenomas (FA) for LOH at 17p13 using 18 probes. Complete clinical follow-up data were available for all patients. We confirmed a high 17p13 LOH rate in FTC (18 of 20) and HCC (13 of 19) and showed an association between 17p13 LOH and advanced tumor grade. Only 4 of 15 PTC and 1 of 7 FA displayed 17p13 LOH. In the HCC we identified a narrow minimal common deleted region between D17S1308 (285 kb from the p-telomer) and D17S695 (696 kb from the p-telomer). This region was flanked centromerically by a breakpoint cluster, further suggesting nonrandom deletion. All but 1 of the PTC and FA with 17p LOH and 50% of the affected FTC also showed LOH in this region. These data suggest that a TSG, involved in HCC pathogenesis, is contained within the D17S1308-D17S695 interval. There are several potential candidate TSGs in this region that are worthy of further study.

Adenocarcinoma, Follicular↗