Partial Methylation Research Articles

1431 EPIGENETIC REGULATION OF MYOPODIN GENE IN PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research VIII1 Apr 20101431 EPIGENETIC REGULATION OF MYOPODIN GENE IN PROSTATE CANCER Temuujin Dansranjavin, Florian Wagenlehner, Alexander Bogumil, Bora Altinkilic, Wolfgang Weidner, Klaus Steger, and Agnieszka Paradowska Temuujin DansranjavinTemuujin Dansranjavin More articles by this author , Florian WagenlehnerFlorian Wagenlehner More articles by this author , Alexander BogumilAlexander Bogumil More articles by this author , Bora AltinkilicBora Altinkilic More articles by this author , Wolfgang WeidnerWolfgang Weidner More articles by this author , Klaus StegerKlaus Steger More articles by this author , and Agnieszka ParadowskaAgnieszka Paradowska More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1124AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Myopodin is an actin binding protein frequently down regulated in prostate cancers (PCa) and PCa derived cell lines. Overexpression of myopodin inhibits PCa growth and metastasis in-vitro and in-vivo. However, less is known about the molecular mechanism underlying the regulation of myopodin gene expression. In this study, we analyzed the involvement of epigenetic mechanisms in PCa-associated regulation of myopodin. METHODS To evaluate the role of epigenetic DNA/histone modifications in the silencing of myopodin gene in PCa, we analyzed the effect of 5-aza-2-deoxycytidine (5-aza-dC) and trichostatin A (TSA) on myopodin mRNA expression in PCa cell lines 22RV1, LNCAP and DU145. Combined bisulfite restriction analysis (COBRA) and bisulfite sequencing of myopodin gene promoter were performed on frozen tissue samples from 37 PCa patients (18 T2, 14 T3, 5 T4) containing 20 of benign prostatic hyperplasia (BPH), 17 of prostate adenocarcinoma. The myopodin mRNA expression levels were examined in relation to methylation status of myopodin gene promoter and clinicopathological parameters of PCa patients. RESULTS All examined PCa cell lines revealed partial methylation of myopodin gene promoter associated with weak (LnCAP) and lack (DU145, 22RV1) of mRNA expression. Myopodin mRNA expression was inducible by TSA. The effect of TSA was enhanced by a combined treatment with 5-aza-dC in DU145 and LNCAP. 59% (10/17) of PCa and 65% (13/20) of BPH revealed a partial methylation of myopodin gene promoter. All matched samples of stromal tissue with PCa (12) and HGPIN with BPH (5) revealed concordant methylation pattern. Among T3-PCa a gradual loss of methylation with increasing dedifferentiation was observed (80% in G2 vs. 44% in G3). CONCLUSIONS Silenced myopodin gene was inducible with TSA and with combined treatment with TSA and 5-aza-dC. These results indicate that epigenetic histone and DNA modifications are involved in PCa associated myopodin inactivation. Partial methylation of myopodin may indicate at histological dedifferentiation in advanced PCa. Giessen, Germany© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e551-e552 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Temuujin Dansranjavin More articles by this author Florian Wagenlehner More articles by this author Alexander Bogumil More articles by this author Bora Altinkilic More articles by this author Wolfgang Weidner More articles by this author Klaus Steger More articles by this author Agnieszka Paradowska More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Methylation Pattern of the IFN-γ Gene in Human Dental Pulp

IntroductionDNA methylation is characterized by the addition of methyl groups in cytosines within cytosine-phosphate-guanine (CpG) islands. Unmethylated islands are related with transcriptionally active structure, whereas methylated DNA recruits methyl-binding proteins that promotes chromatin compaction. Although epigenetic events can influence the expression of cytokines, such events have not been investigated in dental pulp yet. The purpose of the present study was to evaluate the methylation status of the interferon γ (IFN-γ) gene in human dental pulp affected by inflammation compared with pulp tissue of impacted molar teeth and to verify the impact of methylation status in the expression pattern of the gene. MethodsMethylation-specific polymerase chain reaction (MSP) was used to verify the DNA methylation status of the IFN-γ gene in 16 human dental pulps affected by inflammation and in 16 pulp samples of impacted molar teeth. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation, and the expression of IFN-γ was assessed by quantitative real-time PCR (qPCR). ResultsAlthough total methylation was observed in 43.75% of the samples of normal dental pulp tissues, partial methylation or unmethylation was found in 93.75% of the samples of inflamed pulp tissues. All the samples with total methylation in MSP showed no transcription of IFN-γ. The qPCR results showed expression of IFN-γ in 5 of 10 samples with partial methylation. ConclusionThe present study gives the first evidence of the possible participation of epigenetic events in the development of dental pulp inflammation.

P16 promoter methylation in Pb2+-exposed individuals

Background. One of the principle symptoms of lead poisoning is the development of neurological disorders. Neuronal response is closely related to DNA methylation changes. Aim. In this study, we estimated p16 methylation in nine individuals exposed to lead using methylation-specific polymerase chain reaction followed by analysis of the methylated cytosine content of the product by thermal denaturation. Results. We found that, based on lead blood concentration, lead-exposed individuals were divided into two groups. Among highly exposed individuals (blood Pb2+ concentration = 51–100 μg/dL), we observed complete CpG methylation, whereas for low Pb2+ concentrations (blood Pb2+ concentration = 6–11 μg/dL), we observed partial methylation. Conclusion. Our results show that among lead-overexposed individuals, p16 methylation is frequent and extensive, and suggest that DNA methylation could be involved in the mechanism by which lead induces neurotoxicity.

313. EPIGENETIC REGULATION OF THE CRH GENE PROMOTER INVOLVES SPECIFIC CpG DE-METHYLATION

Corticotropin Releasing Hormone (CRH), is expressed in many regions of the central nervous system and in some peripheral tissues, and plays an important role in determining gestational length. In placenta, a cAMP regulatory site (CRE) is crucial for CRH gene regulation. The promoter of CRH gene has 9 CpG sites, which should be the targets of epigenetic regulation by DNA methylation. The BeWo cell line, derived from human gestational choriocarcinoma, has been widely used as an in vitro model for the placenta. BeWo cells only produce CRH after exposure to cAMP. The DNA methyl transferase (DNMT) inhibitor 5-aza-cytidine stimulates CRH expression 5-fold in camp treated BeWo cells, indicating the CRH promoter as a target of DNMTs. To evaluate methylation differences of the 9 CpG sites in CRH gene promoter in BeWo cells after treatment with cAMP. Genomic DNA was extracted from BeWo cells treated or not with cAMP. Sodium bisulfite conversion was used to modify the genomic DNA. PCR was used to amplify the CRH promoter region with primers that did not contain CpG sites. The PCR products were cloned and sequenced. The CpG methylation status of each sample was obtained by comparing the sequencing results with the original sequence. In non-stimulated cells (control) CpG -4 was methylated in 50% of the clones and CpG -6 was methylated in 75% of the clones, but the other 7 sites were methylated in every clone. In the cAMP treated cells however there was 100% methylation at CpG sites 6 through 9, but only partial methylation at CpG-1 and 3 (60%), CpG-4 and 5 (40%). Most interestingly, there was no methylation found at CpG-2 in any of the clones from cAMP treated cells, indicating that specific CpG de-methylation around the CRE is required for CRH gene expression.

Prospects for epigenetic research within cohort studies of psychological disorder: A pilot investigation of a peripheral cell marker of epigenetic risk for depression

Cohort studies have considerable prima facie value for investigating epigenetic processes in psychological disorder; however, the future prospects for such studies will depend on valid peripheral markers. The purpose of this pilot study was to investigate association between buccal cell methylation and risk for depression. Epigenotyping was limited to promoter methylation of the serotonin transporter gene ( 5HTT) . A transcription limiting VNTR in the 5HTT promoter ( 5HTTLPR) was also genotyped. A nested sample of 25 depressed and 125 non-depressed adolescents was drawn from an established longitudinal study of adolescent health. There was no association between depressive symptoms and either buccal cell 5HTT methylation or 5HTTLPR. However, depressive symptoms were more common among those with elevated buccal cell 5HTT methylation who carried 5HTTLPR short-allele (OR 4.9, CI 1.9–13, p = 0.001). Both complete and partial (as little as 10%) methylation of a 5HTT reporter gene in an expressing cell line reduced 5HTT activity. Replication is needed.

HTERT methylation and expression in gastric cancer

Gastric cancer is the second most prevalent cause of cancer death worldwide. DNA methylation is a common event in gastric carcinogenesis. hTERT seems to be the rate-limiting determinant of telomerase activation, which is responsible for stability and life span. hTERT hypermethylation has been associated with telomerase expression. In the present study, we investigated the promoter methylation status and hTERT protein expression in gastric cancer and normal mucosa samples. One hundred and nine gastric cancer and 53 normal mucosa samples were investigated through methylation-specific PCR. Immunohistochemistry was analysed using peroxidase in 55 gastric cancer and 18 normal gastric mucosa samples. This is the first study evaluating hTERT methylation status in gastric carcinogenesis. We did not observe hTERT protein expression in normal gastric mucosa. Moreover, hTERT expression was observed in 80% of tumours and was associated with gastric cancer (p < 0.0001). Partial methylation was the most frequent pattern in gastric samples, even in normal mucosa. The frequency of specimens presenting hypermethylation was significantly higher in tumours than in normal mucosa samples (p = 0.0002), although the presence of hypermethylated promoter was not associated with a higher frequency of hTERT expression. A low correlation between hTERT protein expression and methylation was verified in gastric cancer samples. There was a clear difference in the frequency of hTERT expression and methylation within tumoral and non-tumoral tissues. Methylation status and telomerase expression may be useful for the diagnosis of gastric cancer and may have an impact on the anti-telomerase strategy for cancer therapy.

Abstract A8: Role of the WWOX gene in non-small cell lung cancer

Abstract Lung cancer is the most common cause of cancer-related deaths worldwide and like many other cancers is affected by different genetic, epigenetic and environmental factors. The WWOX gene is a tumor supressor gene located on 16q23.3–24.1. It has been shown that it loses its function due to differences in genetic and epigenetic mechanisms. In this study we investigated whether the presence of WWOX gene mutations in lung cancer. The entire coding exons and flanking intronic sequences of the gene were analysed by PCR amplification from the genomic DNA and direct sequencing in 50 patients with non-small cell lung cancer (NSCLC). WWOX gene mutations were detected in five out of 50 primary tumors (10%). One of these mutations was an insertion in exon 4 at the third nucleotide of codon 120. This mutation was observed in 2 patients and leads to a frameshift creating a stop codon at codon 133. The second mutation was an insertion at position 2583 which also causes a shift in the reading frame. Finally, heterozygous C→G transitions on position 2807 at the first nucleotide of codon 282 (exon 8) were identified in 2 patients. This mutation affecting codon 282 leads to the substitution of alanine instead of proline. We also observed a previously defined G to A polymorphism at the nucleotide 2196 in homozygous and heterozygous form in 20 and 5 patients, respectively. Additionally we detected 12 different intronic substitutions. In addition to inactivating mutations target genes may also be silenced epigenetically. Therefore, besides mutation screening we also analyzed the methylation profile of the promoter region of the WWOX gene in tumor tissue and adjacent non-cancerous tissue samples by bisulphite modification and methylation-specific PCR. Promoter methylation was detected in 76% of the tumor tissue samples. While 20% of the samples displayed partial methylation 56% were completely methylated. In the adjacent normal tissue samples partial and complete methylation frequencies were 6% and 58%, respectively. Methylation frequency was significantly higher in the tumor tissue samples than in normal tissue samples (p=0.014). The high frequency of mutations and epigenetic modifications suggest that the WWOX gene may play a significant role in the etiology of NSCLC. Citation Information: Cancer Res 2009;69(23 Suppl):A8.

Abstract C20: MGMT promoter methylation and analysis of microsatellite instability in head and neck cancer by consensus markers

Abstract Head and neck cancer is the sixth most common cancer in the world and traditional prognostic indices are of limited use in predicting prognosis. Therefore, there is a need for a beter understanding of the molecular pathogenesis of the disease and a great effort is being made to determine new prognostic and predictive markers that could improve stratification of the patients Polymorphic microsatellites are useful genetic markers and important tools for studying genetic alterations in tumors. Microsatellite instability refers to the impairment of the mismatch repair system resulting in gains or losses in the number of the tandem repeat units. Cells with mutated mismatch repair genes are not able to correctly repair these errors. The importance of microsatellite instability in head and neck cancer is not well established because of lack of comparable parameters, use of different markers, different criteria in classification and discrepancies concerning the loci examined. Recently it has been shown that mononucleotide markers are more sensitive and specific than dinucleotide markers for the detection of microsatelite instability. No standard approach for the study of MSI has been agreed to in HNC. In this study we analyzed the role and incidence of microsatellite instability in head and neck cancer with criteria and markers recommended by NCI which have been widely used for different types of cancer. We also analyzed methylation of the promoter region of the MGMT gene which functions in the repair pathway of alkylating nitrosocompounds. Microsatellite instability was analyzed in matching tumor and normal tissue samples from 99 patients with head and neck cancer using a panel of five (NR21, BAT25, BAT26, NR24 and MONO27) mononucletoide markers. Two pentanucleotide markers were used to correctly identify the individual tumor and normal tissue samples. The analyses were performed on an ABI Prism 310 capillary electrophoresis system. Microsatellite instability was observed in 26 patients (26.2%). The most frequently affected markers were BAT 25 and NR21 which displayed changes in repeat length in 19 and 11 patients, respectively. Among the microsatellite-positive patients 17 tumor samples (17.1%) displayed high degrees of instability while the remaining 9 samples were instable in only one locus. In five patients instability was observed in three or more loci. Instability was more frequent in tumors with advanced stages. Significant methylation of the MGMT gene was observed in most patients. In 31 patients both alleles were methylated, while in 65 samples partial methylation was observed. Citation Information: Cancer Res 2009;69(23 Suppl):C20.

Aberrant DNA Methylation and Transcriptional Silencing of DLC-1 in Waldenstrom's Macroglobulinemia.

Abstract 2954Poster Board II-930The deleted in liver cancer-1 (DLC-1) gene encodes a Rho GTPase activating protein (RhoGAP) with potential tumor-suppressor activity. Hypermethylation in the DLC-1 promoter is an aberrant epigenetic modification associated with transcriptional silencing of DLC-1 in various types of human cancers. To explore the epigenetic alteration of DLC-1 in Waldenström's macroglobulinemia (WM), we investigated the methylation status of the DLC-1 promoter and its correlation with DLC-1 mRNA expression in cell lines and primary WM patient samples. Initial analysis using methylation-specific PCR (MSP) showed that DLC-1 promoter was completely methylated in WM-WSU and partially methylated in BCWM.1. Using quantitative RT-PCR, DLC-1 mRNA expression was detectable in BCWM.1, but not WM-WSU. These results suggested a correlation between the DLC-1 methylation status and mRNA expression. Similarly, among multiple myeloma (MM) cell lines, we showed that RPMI and U266 exhibited complete methylation, whereas INA6 showed partial methylation, and no methylation was detectable in MM1S and MM1R cells. Similar in WM cells, DLC-1 methylation status was highly correlated with the mRNA expression in these MM cell lines. Of 37 WM patient samples examined, 24 (65%) exhibited methylation in the DLC-1 promoter. In contrast, no methylation of DLC-1 was observed in 4 healthy volunteers. The methylation status was further confirmed using bisulfite DNA sequencing in a subset of WM patients. Quantitative RT-PCR analysis showed that DLC-1 mRNA expression was significantly lower in WM patients compared to healthy volunteers (p=0.001). Treatment with demethylation agents azacytidine or 5-aza-deoxycytidine resulted in significant reactivation of DLC-1 transcription in the WM cell lines WM-WSU and BCWM.1. In addition, a synergistic induction of DLC-1 transcription was observed in the presence of azacytidine and the HDAC inhibitor Vorinostat in BCWM.1 and primary WM patient cells. Moreover, functional studies showed that overexpression of DLC-1 induced cell growth arrest and apoptosis in BCWM.1. DLC-1 methylation status was also correlated with serum sCD27 levels in WM patients (p=0.004), which is secreted by WM cells, serves as a marker of disease burden and facilitates CD40L directed paracrine stimulation by mast cells. Taken together, these results suggest that the down-regulation of DLC-1 via aberrant DNA methylation plays a role in the pathogenesis of WM, and represents a novel therapeutic target in the treatment of WM. Disclosures:No relevant conflicts of interest to declare.

Hypermethylation Leads to Down-regulated 14-3-3 Sigma Expression in Nasopharyngeal Carcinoma Tissues*

In order to explore the role of 14-3-3σ gene methylation in nasopharyngeal carcinoma pathogenesis, biopsy specimens of 75 nasopharyngeal carcinoma tissues and 25 normal nasopharyngeal mucosal tissues were harvested for research. Then, methylation specific PCR (MSP) experiment was introduced to reveal 14-3-3σ gene methylation status of tissues, and reverse transcriptional PCR (RT-PCR) assay was employed to quantify the expression of 14-3-3σ mRNA, further more, immunohistochemical staining was undertaken to assess the 14-3-3σ protein expression level. The MSP experiment results indicate 14-3-3σ gene methylation status in nasopharyngeal carcinoma tissues was 4 exhaustive methylation, 59 partial methylation and 12 unmethylation, while in normal nasopharyngeal mucosal tissues was 7 partial methylation and 18 unmethylation. The frequency of 14-3-3σ methylation in nasopharyngeal carcinoma tissues was significantly higher than that in normal nasopharyngeal mucosal tissues (84% vs 28% , χ2 =28; P 0.05). The results of RT-PCR and immunohistochemical staining discovered that 14-3-3σ gene exhaustive methylation leads to absent 14-3-3σ mRNA as well as protein expression in nasopharyngeal carcinoma tissues and normal nasopharyngeal mucosal tissues. Tissues characterized with partial methylation 14-3-3σ gene represented remarkable down-regulated 14-3-3σ mRNA and protein expression level when compared with tissues characterized with unmethylation 14-3-3σ gene. Graded correlation analysis discovered that 14-3-3σ gene hypermethylation status is positively correlated with NPC lymphoid node metastasis and NPC clinical staging. The research demonstrated that 14-3-3σ gene was frequently hypermethylated in nasopharyngeal carcinoma tissues and leads to decreased or depleted gene expression, 14-3-3σ expression level was correlated to nasopharyngeal carcinoma lymphoid node metastasis and NPC clinical staging.

Epigenetic control of the ubiquitin carboxyl terminal hydrolase 1 in renal cell carcinoma

BackgroundThe ubiquitin carboxyl-terminal hydrolase 1 (UCHL1) gene involved in the regulation of cellular ubiquitin levels plays an important role in different cellular processes including cell growth and differentiation. Aberrant expression of UCHL1 has been found in a number of human solid tumors including renal cell carcinoma (RCC). In RCC, UCHL1 overexpression is associated with tumor progression and an altered von Hippel Lindau gene expression.MethodsTo determine the underlying mechanisms for the heterogeneous UCHL1 expression pattern in RCC the UCHL1 promoter DNA methylation status was determined in 17 RCC cell lines as well as in 32 RCC lesions and corresponding tumor adjacent kidney epithelium using combined bisulfite restriction analysis as well as bisulfite DNA sequencing.ResultsUCHL1 expression was found in all 32 tumor adjacent kidney epithelium samples. However, the lack of or reduced UCHL1 mRNA and/or protein expression was detected in 13/32 RCC biopsies and 7/17 RCC cell lines and due to either a total or partial methylation of the UCHL1 promoter DNA. Upon 2'-deoxy-5-azacytidine treatment an induction of UCHL1 mRNA and protein expression was found in 9/17 RCC cell lines, which was linked to the demethylation degree of the UCHL1 promoter DNA.ConclusionPromoter hypermethylation represents a mechanism for the silencing of the UCHL1 gene expression in RCC and supports the concept of an epigenetic control for the expression of UCHL1 during disease progression.

DNA cleavage and methylation specificity of the single polypeptide restriction–modification enzyme LlaGI

LlaGI is a single polypeptide restriction–modification enzyme encoded on the naturally-occurring plasmid pEW104 isolated from Lactococcus lactis ssp. cremoris W10. Bioinformatics analysis suggests that the enzyme contains domains characteristic of an mrr endonuclease, a superfamily 2 DNA helicase and a γ-family adenine methyltransferase. LlaGI was expressed and purified from a recombinant clone and its properties characterised. An asymmetric recognition sequence was identified, 5′-CTnGAyG-3′ (where n is A, G, C or T and y is C or T). Methylation of the recognition site occurred on only one strand (the non-degenerate dA residue of 5′-CrTCnAG-3′ being methylated at the N6 position). Double strand DNA breaks at distant, random sites were only observed when two head-to-head oriented, unmethylated copies of the site were present; single sites or pairs in tail-to-tail or head-to-tail repeat only supported a DNA nicking activity. dsDNA nuclease activity was dependent upon the presence of ATP or dATP. Our results are consistent with a directional long-range communication mechanism that is necessitated by the partial site methylation. In the accompanying manuscript [Smith et al. (2009) The single polypeptide restriction–modification enzyme LlaGI is a self-contained molecular motor that translocates DNA loops], we demonstrate that this communication is via 1-dimensional DNA loop translocation. On the basis of this data and that in the third accompanying manuscript [Smith et al. (2009) An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI], we propose that LlaGI is the prototype of a new sub-classification of Restriction-Modification enzymes, named Type I SP (for Single Polypeptide).

Geographic variation and environmental conditions as cofactors in Chlamydia psittaci association with ocular adnexal lymphomas: a comparison between Italian and African samples

A particular extra-nodal lymphoma type arises from B cells of the marginal zone (MZ) of mucosa-associated lymphoid tissue (MALT). The aetiology of MZ lymphomas suggests that they are associated with chronic antigenic stimulation by microbial pathogens, among which Helicobacter pylori-associated gastric MALT lymphoma is the best studied. Recently, MALT lymphomas have been described in the context of chronic conjunctivitis, which can be associated with Chlamydia spp. infection. Studies from Italy showed the presence of Chlamydia psittaci in 87% of ocular adnexal lymphomas (OAL), and C. psittaci has been described in a large part of samples from Austria and Korea as well. However, this finding was not always confirmed by other studies, suggesting that the association with C. psittaci may depend on geographic heterogeneity. Interestingly, none of the studies up to now has been carried out in the African population, where a strong association between infectious agents and the occurrence of human neoplasms has been reported. This study was designed to investigate the possible association of Chlamydia psittaci in cases retrieved from Kenya, compared to cases from Italy. Our results showed that there was a marked variation between the two geographical areas in terms of association with C. psittaci, as 17% (5/30) of the samples from Italy were positive for C. psittaci, whereas no association with this pathogen was observed in any of the African samples (0/9), suggesting that other cofactors may determine the OAL occurrence in those areas. OAL cases are often characterized by down-regulation of p16/INK4a expression and promoter hypermethylation of the p16/INK4a gene. Our results showed a partial methylation of p16/INK4a promoter in C. psittaci-negative cases, whereas no hypermethylation of this gene was found in C. psittaci-positive cases, suggesting that mechanisms other than promoter hypermethylation lead to p16/INK4a silencing in C. psittaci-positive cases. We may conclude that the role of epidemiologic, environmental and genetic factors, must be considered in the aetiology of this disease.

12LBA A Plasma-based colorectal cancer (CRC) screening assay using DNA methylation markers - first results of multicenter studies

Background & Aims: Methylation of the hMLH1 promoter region has been suggested to cause microsatellite instability (MSI) in sporadic colorectal carcinoma (CRC). We studied the methylation profile in a wide region of the hMLH1 promoter and compared with the hMLH1 protein expression and MSI status in 88 cases of sporadic CRC. Methods: Na-bisulfite treatment and polymerase chain reaction single-strand conformation polymorphism analysis was performed using 5 sets of polymerase chain reaction primers spanning the promoter region of the hMLH1 to examine methylation status. Results were compared with immunostaining using anti-hMLH1 monoclonal antibody and MSI status of the tumor samples. Results: Methylation status was classified as full or partial methylation. Full methylation indicates the methylation of all CpG sites in the examined regions. Methylation of the hMLH1 promoter was observed in 88.9% (16 of 18) of CRCs showing high frequency MSI (MSI-H), among which 89% (14 of 16) had full methylation with reduced hMLH1 protein expression. All cases showing full methylation were proximal colon tumors with MSI-H. In cases with partial methylation, only the upstream region of the hMLH1 promoter was methylated. Partial methylation was also shown in 33.3% (6 of 18) of the normal mucosa of MSI-H cases. Frequencies of methylation were significantly correlated with female gender (P = 0.0009) and aging (P = 0.007). Conclusions: Full methylation of the hMLH1 promoter region and subsequent gene inactivation may play a crucial role in the carcinogenesis of MSI-H CRCs in the proximal colon. Methylation upstream of the hMLH1 promoter appears to be an early event in the carcinogenesis of MSI-H tumors.GASTROENTEROLOGY 2001;121:1300-1309

The effect of particle shape and hydrophobicity in flotation

The effect of particle shape on the flotation process has been investigated in laboratory experiments with monosized spherical ballotini and ground ballotini. The particles were treated by partial methylation with trimethylchlorosilane to achieve varying degrees of hydrophobicity. In flotation, the process of film thinning and liquid drainage is critical in the formation of stable bubble–particle attachments and this is affected by the particle shape and surface hydrophobicity. Flotation tests with different particle sizes were conducted in a modified batch Denver cell. Predictions from a computational fluid dynamic model of the flotation cell that incorporates fundamental aspects of bubble–particle attachment were compared with data from flotation tests. Contact angles of the particles were measured using a capillary rise technique to indicate surface hydrophobicity. Ground ballotini generally has higher flotation rates than spherical ballotini; the results are consistent with effects from faster film thinning and rupture at rough surfaces and are well correlated by the sphericity index.

High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing

DNA methylation stabilizes developmentally programmed gene expression states. Aberrant methylation is associated with disease progression and is a common feature of cancer genomes. Presently, few methods enable quantitative, large-scale, single-base resolution mapping of DNA methylation states in desired regions of a complex mammalian genome. Here, we present an approach that combines array-based hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in genomic regions spanning hundreds of thousands of bases. This single molecule strategy enables methylation variable positions to be quantitatively examined with high sampling precision. Using bisulfite capture, we assessed methylation patterns across 324 randomly selected CpG islands (CGI) representing more than 25,000 CpG sites. A single lane of Illumina sequencing permitted methylation states to be definitively called for >90% of target sties. The accuracy of the hybrid-selection approach was verified using conventional bisulfite capillary sequencing of cloned PCR products amplified from a subset of the selected regions. This confirmed that even partially methylated states could be successfully called. A comparison of human primary and cancer cells revealed multiple differentially methylated regions. More than 25% of islands showed complex methylation patterns either with partial methylation states defining the entire CGI or with contrasting methylation states appearing in specific regional blocks within the island. We observed that transitions in methylation state often correlate with genomic landmarks, including transcriptional start sites and intron-exon junctions. Methylation, along with specific histone marks, was enriched in exonic regions, suggesting that chromatin states can foreshadow the content of mature mRNAs.

Ras pathway activation in hepatocellular carcinoma and anti-tumoral effect of combined sorafenib and rapamycin in vivo

The success of sorafenib in the treatment of advanced hepatocellular carcinoma (HCC) has focused interest on the role of Ras signaling in this malignancy. We investigated the molecular alterations of the Ras pathway in HCC and the antineoplastic effects of sorafenib in combination with rapamycin, an inhibitor of mTOR pathway, in experimental models. Gene expression (qRT-PCR, oligonucleotide microarray), DNA copy number changes (SNP-array), methylation of tumor suppressor genes (methylation-specific PCR) and protein activation (immunohistochemistry) were analysed in 351 samples. Anti-tumoral effects of combined therapy targeting the Ras and mTOR pathways were evaluated in cell lines and HCC xenografts. Different mechanisms accounted for Ras pathway activation in HCC. H-ras was up-regulated during different steps of hepatocarcinogenesis. B-raf was overexpressed in advanced tumors and its expression was associated with genomic amplification. Partial methylation of RASSF1A and NORE1A was detected in 89% and 44% of tumors respectively, and complete methylation was found in 11 and 4% of HCCs. Activation of the pathway (pERK immunostaining) was identified in 10.3% of HCC. Blockade of Ras and mTOR pathways with sorafenib and rapamycin reduced cell proliferation and induced apoptosis in cell lines. In vivo, the combination of both compounds enhanced tumor necrosis and ulceration when compared with sorafenib alone. Ras activation results from several molecular alterations, such as methylation of tumor suppressors and amplification of oncogenes (B-raf). Sorafenib blocks signaling and synergizes with rapamycin in vivo, preventing tumor progression. These data provide the rationale for testing this combination in clinical studies.

LSAMP, a novel candidate tumor suppressor gene in human osteosarcomas, identified by array comparative genomic hybridization

Osteosarcomas are the most common primary malignant tumor of bone, and almost all conventional osteosarcomas are high-grade tumors with complex karyotypes. We have examined DNA copy number changes in 36 osteosarcoma tumors and 20 cell lines using microarray-based comparative genomic hybridization. The most frequent minimal recurrent regions of gain identified in the tumor samples were in 1q21.2-q21.3 (78% of the samples), 1q21.3-q22 (78%), and 8q22.1 (72%). Minimal recurrent regions in 10q22.1-q22.2 (81%), 6q16.1 (67%), 13q14.2 (67%), and 13q21.1 (67%) were most frequently lost. A small region in 3q13.31 (2.1 Mb) containing the gene limbic system-associated membrane protein (LSAMP) was frequently deleted (56%). LSAMP has previously been reported to be a candidate tumor suppressor gene in other cancer types. The deletion was validated using fluorescence in situ hybridization, and the expression level and promoter methylation status of LSAMP were investigated using quantitative real-time reverse transcription PCR and methylation-specific PCR, respectively. LSAMP showed low expression compared to two normal bone samples in 6/15 tumors and 5/9 cell lines with deletion of 3q13.31, and also in 5/14 tumors and 3/11 cell lines with normal copy number or gain. Partial or full methylation of the investigated CpG island was identified in 3/30 tumors and 7/20 cell lines. Statistical analyses revealed that loss of 11p15.4-p15.3 and low expression of LSAMP (both P = 0.011) were significantly associated with poor survival. Our results show that LSAMP is a novel candidate tumor suppressor gene in osteosarcomas.

Encapsulation of Small Ionic Molecules within α-Cyclodextrins

Results from molecular dynamics experiments pertaining to the encapsulation of ClO4- within the hydrophobic cavity of an aqueous alpha-cyclodextrin (alpha-CD) are presented. Using a biased sampling procedure, we constructed the Gibbs free energy profile associated with the complexation process. The profile presents a global minimum at the vicinity of the primary hydroxyl groups, where the ion remains tightly coordinated to four water molecules via hydrogen bonds. Our estimate for the global free energy of encapsulation yields DeltaGenc approximately -2.5 kBT. The decomposition of the average forces acting on the trapped ion reveals that the encapsulation is controlled by Coulomb interactions between the ion and OH groups in the CD, with a much smaller contribution from the solvent molecules. Changes in the previous results, arising from the partial methylation of the host CD and modifications in the charge distribution of the guest molecule are also discussed. The global picture that emerges from our results suggests that the stability of the ClO4- encapsulation involves not only the individual ion but also its first solvation shell.

Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles.

Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus 1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns and trans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionally silenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while its transcriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in early Lo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue in the parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgenes were silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquired partial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similar epigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate, and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus. Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated with dedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.