Promoter methylation and expression of MGMT and the DNA mismatch repair genes MLH1, MSH2, MSH6 and PMS2 in paired primary and recurrent glioblastomas.

PubMed

Felsberg, Jörg; Thon, Niklas; Eigenbrod, Sabina; Hentschel, Bettina; Sabel, Michael C; Westphal, Manfred; Schackert, Gabriele; Kreth, Friedrich Wilhelm; Pietsch, Torsten; Löffler, Markus; Weller, Michael; Reifenberger, Guido; Tonn, Jörg C

2011-08-01

Epigenetic silencing of the O(6) -methylguanine-DNA methyltransferase (MGMT) gene promoter is associated with prolonged survival in glioblastoma patients treated with temozolomide (TMZ). We investigated whether glioblastoma recurrence is associated with changes in the promoter methylation status and the expression of MGMT and the DNA mismatch repair (MMR) genes MLH1, MSH2, MSH6 and PMS2 in pairs of primary and recurrent glioblastomas of 80 patients, including 64 patients treated with radiotherapy and TMZ after the first operation. Among the primary tumors, the MGMT promoter was methylated in 31 patients and unmethylated in 49 patients. In 71 patients (89%), the MGMT promoter methylation status of the primary tumor was retained at recurrence. MGMT promoter methylation, but not MGMT protein expression, was associated with longer progression-free survival, overall survival and postrecurrence survival (PRS). Moreover, PRS was increased under salvage chemotherapy. Investigation of primary and recurrent glioblastomas of 43 patients did not identify promoter methylation in any of the four MMR genes. However, recurrent glioblastomas demonstrated significantly lower MSH2, MSH6 and PMS2 protein expression as detected by immunohistochemistry. In conclusion, reduced expression of MMR proteins, but not changes in MGMT promoter methylation, is characteristic of glioblastomas recurring after the current standards of care. Copyright © 2011 UICC.

O6-Methylguanine-DNA methyltransferase protein expression by immunohistochemistry in brain and non-brain systemic tumours: systematic review and meta-analysis of correlation with methylation-specific polymerase chain reaction.

PubMed

Brell, Marta; Ibáñez, Javier; Tortosa, Avelina

2011-01-26

The DNA repair protein O6-Methylguanine-DNA methyltransferase (MGMT) confers resistance to alkylating agents. Several methods have been applied to its analysis, with methylation-specific polymerase chain reaction (MSP) the most commonly used for promoter methylation study, while immunohistochemistry (IHC) has become the most frequently used for the detection of MGMT protein expression. Agreement on the best and most reliable technique for evaluating MGMT status remains unsettled. The aim of this study was to perform a systematic review and meta-analysis of the correlation between IHC and MSP. A computer-aided search of MEDLINE (1950-October 2009), EBSCO (1966-October 2009) and EMBASE (1974-October 2009) was performed for relevant publications. Studies meeting inclusion criteria were those comparing MGMT protein expression by IHC with MGMT promoter methylation by MSP in the same cohort of patients. Methodological quality was assessed by using the QUADAS and STARD instruments. Previously published guidelines were followed for meta-analysis performance. Of 254 studies identified as eligible for full-text review, 52 (20.5%) met the inclusion criteria. The review showed that results of MGMT protein expression by IHC are not in close agreement with those obtained with MSP. Moreover, type of tumour (primary brain tumour vs others) was an independent covariate of accuracy estimates in the meta-regression analysis beyond the cut-off value. Protein expression assessed by IHC alone fails to reflect the promoter methylation status of MGMT. Thus, in attempts at clinical diagnosis the two methods seem to select different groups of patients and should not be used interchangeably.

Quantitative analysis of DNA methylation in the promoter region of the methylguanine-O(6) -DNA-methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis.

PubMed

Goedecke, Simon; Mühlisch, Jörg; Hempel, Georg; Frühwald, Michael C; Wünsch, Bernhard

2015-12-01

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine-O(6) -DNA-Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non-methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non-linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

O6-methylguanine-DNA methyltransferase activity in human buccal mucosal tissue and cell cultures. Complex mixtures related to habitual use of tobacco and betel quid inhibit the activity in vitro.

PubMed

Liu, Y; Egyhazi, S; Hansson, J; Bhide, S V; Kulkarni, P S; Grafström, R C

1997-10-01

Extracts prepared from tissue specimens of normal, non-tumourous human buccal mucosa, and cultured buccal epithelial cells and fibroblasts, exhibited O6-methylguanine-DNA methyltransferase (MGMT) activity by catalysing the repair of the premutagenic O6-methylguanine lesion in isolated DNA with rates of 0.2 to 0.3 pmol/mg protein. An SV40 T antigen-immortalized buccal epithelial cell line termed SVpgC2a and a buccal squamous carcinoma line termed SqCC/Y1, both of which lack normal tumour suppressor gene p53 function, exhibited about 50 and 10% of the MGMT activity of normal cells, respectively. The normal, experimentally transformed and tumourous buccal cell types showed MGMT mRNA levels which correlated with their respective levels of MGMT activity. Exposure of buccal cell cultures to various organic or water-based extracts of products related to the use of tobacco and betel quid, decreased both cell survival (measured by reduction of tetrazolium dye) and MGMT activity (measured subsequently to the exposures in cellular extracts). Organic extracts of bidi smoke condensate and betel leaf showed higher potency than those of tobacco and snuff. An aqueous snuff extract also decreased both parameters, whereas an aqueous areca nut extract was without effect. The well-established sulph-hydryl-reactive agent Hg2+, a corrosion product of dental amalgam, served as a positive control and decreased MGMT activity following treatment of cells within a range of 1-10 microM. Taken together, significant MGMT activities were demonstrated in buccal tissue specimens and in the major buccal mucosal cell types in vitro. Lower than normal MGMT activity in two transformed buccal epithelial cell lines correlated with decreased MGMT mRNA and lack of functional p53. Finally, in vitro experiments suggested the potential inhibition of buccal mucosal MGMT activity by complex mixtures present in the saliva of tobacco and betel nut chewers.

Prognostic significance of O6-methylguanine-DNA methyltransferase protein expression in patients with recurrent glioblastoma treated with temozolomide.

PubMed

Nagane, Motoo; Kobayashi, Keiichi; Ohnishi, Akiko; Shimizu, Saki; Shiokawa, Yoshiaki

2007-12-01

Temozolomide (TMZ) is active against newly diagnosed glioblastoma (GBM), and O(6)-methylguanine-DNA methyltransferase (MGMT) is implicated in resistance to TMZ and nitrosoureas. We evaluated the efficacy and safety of the standard 5-day TMZ regimen in patients with recurrent GBM after initial therapy including nitrosourea-based chemotherapy, in conjunction with an analysis of the prognostic value of MGMT protein expression regarding response to TMZ and survival. From September 2003 to January 2007, 30 patients having recurrent GBM received 150-200 mg/m(2)/day of TMZ for five consecutive days every 28 days. Tumor tissue from 19 patients was analysed for MGMT protein expression using western blotting, and 17 of them were assessable for a response. The overall response rate was 23.5% (one complete response and three partial responses). Six patients had stable disease (35.3%). Median progression-free survival (PFS) time was 2.2 months, and median overall survival (OS) time was 9.9 months from the initiation of TMZ therapy. Patients with low MGMT protein expression had a significantly improved PFS (P = 0.016) and OS (P = 0.019) compared to those with high expression. Both low MGMT expression (P = 0.040) and re-resection at relapse (P = 0.014) persisted as significant independent favorable prognostic factors for OS. The most common grade 3 and 4 hematological toxicity was lymphopenia (22.2%). The standard 5-day TMZ regimen resulted in moderate antitumor activity with an acceptable safety profile in patients with nitrosourea-pretreated recurrent GBM, and protein expression of MGMT is an important prognostic factor for patients treated with TMZ even after recurrence.

Enhanced in vivo selection of bone marrow cells by retroviral-mediated coexpression of mutant O6-methylguanine-DNA-methyltransferase and HOXB4.

PubMed

Milsom, Michael D; Woolford, Lorna B; Margison, Geoffrey P; Humphries, R Keith; Fairbairn, Leslie J

2004-11-01

To attain therapeutic levels of gene-modified hematopoietic stem cells, it may be necessary in the majority of disorders to provide an in vivo selective advantage that facilitates the expansion of their numbers. A popular strategy to achieve in vivo selection has been to employ drug selection while coexpressing a transgene that conveys chemoresistance, such as O6-methylguanine-DNA-methyltransferase (MGMT). An alternate approach is to confer an enhanced proliferative potential upon gene-modified hematopoietic stem cells through the delivery of the homeobox transcription factor HOXB4. By developing a novel tricistronic retroviral vector, we have facilitated the simultaneous coexpression of a mutant version of MGMT and HOXB4 in retrovirally transduced bone marrow. Using an in vivo competitive repopulation assay, we demonstrate that primary bone marrow cells containing this construct show enhanced reconstitution following transplant and improved selection subsequent to chemotherapeutic challenge in comparison to cells expressing either HOXB4 or MGMT alone. This selection advantage was evident even when HOXB4/MGMT-coexpressing cells were infused along with a large excess of unmodified cells. We propose that this selection cassette may facilitate the in vivo expansion of gene-modified hematopoietic stem cells at a level in excess of previous strategies.

Implication of a chromosome 15q15.2 locus in regulating UBR1 and predisposing smokers to MGMT methylation in lung

PubMed Central

Leng, Shuguang; Wu, Guodong; Collins, Leonard B.; Thomas, Cynthia L.; Tellez, Carmen S.; Jauregui, Andrew R.; Picchi, Maria A.; Zhang, Xiequn; Juri, Daniel E.; Desai, Dhimant; Amin, Shantu G.; Crowell, Richard E.; Stidley, Christine A.; Liu, Yushi; Swenberg, James A.; Lin, Yong; Wathelet, Marc G.; Gilliland, Frank D.; Belinsky, Steven A.

2015-01-01

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. A single nucleotide polymorphism (SNP) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells (HBEC), while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells. PMID:26183928

MGMT methylation: a marker of response to temozolomide in low-grade gliomas.

PubMed

Everhard, Sibille; Kaloshi, Gentian; Crinière, Emmanuelle; Benouaich-Amiel, Alexandra; Lejeune, Julie; Marie, Yannick; Sanson, Marc; Kujas, Michèle; Mokhtari, Karima; Hoang-Xuan, Khê; Delattre, Jean-Yves; Thillet, Joëlle

2006-12-01

The methylation status of the O6-methylguanine-methyltransferase promoter (MGMTP) was evaluated in 68 low-grade gliomas treated by neoadjuvant temozolomide. Methylated MGMTP was detected in 63 of 68 (92.6 %) patients and was a favorable predictor of progression-free survival as compared with unmethylated MGMTP tumors (p < 0.0001). Assessment of MGMTP status could help identifying low-grade gliomas patients more likely to respond to chemotherapy or to benefit from MGMT depletion strategies.

The MGMT promoter SNP rs16906252 is a risk factor for MGMT methylation in glioblastoma and is predictive of response to temozolomide.

PubMed

Rapkins, Robert W; Wang, Fan; Nguyen, HuyTram N; Cloughesy, Timothy F; Lai, Albert; Ha, Wendy; Nowak, Anna K; Hitchins, Megan P; McDonald, Kerrie L

2015-12-01

Promoter methylation of O(6)-methylguanine-DNA methyltransferase (MGMT) is an important predictive biomarker in glioblastoma. The T variant of the MGMT promoter-enhancer single nucleotide polymorphism (SNP; rs16906252) has been associated with the presence of MGMT promoter methylation in other cancers. We examined the association of the T allele of rs16906252 with glioblastoma development, tumor MGMT methylation, MGMT protein expression, and survival outcomes. Two independent temozolomide-treated glioblastoma cohorts-one Australian (Australian Genomics and Clinical Outcomes of Glioma, n = 163) and the other American (University of California Los Angeles/Kaiser Permanente Los Angeles, n = 159)-were studied. Allelic bisulphite sequencing was used to determine if methylation was specific to the T allele. Additionally, we compared the incidence of the T allele between glioblastoma cases and matched controls to assess whether it was a risk factor for developing MGMT methylated glioblastoma. Carriage of the T allele of the rs16906252 SNP was associated with both MGMT methylation and low MGMT protein expression and predicted significantly longer survival in temozolomide-treated patients with both MGMT methylated and nonmethylated glioblastoma. Methylation was linked to the T allele, inferring that the T variant plays a key role in the acquisition of MGMT methylation. Carriage of the T allele was associated with a significantly elevated risk of developing glioblastoma (adjusted odds ratio, 1.96; P = .013), increasing further when glioblastoma was classified by the presence of MGMT methylation (adjusted odds ratio, 2.86; P = .001). The T allele of the rs16906252 SNP is a key determinant in the acquisition of MGMT methylation in glioblastoma. Temozolomide-treated patients with the rs16906252 T genotype have better survival, irrespective of tumor methylation status. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All

Comprehensive Analysis of MGMT Promoter Methylation: Correlation with MGMT Expression and Clinical Response in GBM

PubMed Central

Shah, Nameeta; Lin, Biaoyang; Sibenaller, Zita; Ryken, Timothy; Lee, Hwahyung; Yoon, Jae-Geun; Rostad, Steven; Foltz, Greg

2011-01-01

O6-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089–13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301–7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting. PMID

Quantitation of O6-methylguanine-DNA methyltransferase gene messenger RNA in gliomas by means of real-time RT-PCR and clinical response to nitrosoureas.

PubMed

Tanaka, Satoshi; Oka, Hidehiro; Fujii, Kiyotaka; Watanabe, Kaoru; Nagao, Kumi; Kakimoto, Atsushi

2005-09-01

1. O6-methylguanine-DNA methyltransferase (MGMT) mRNA was measured in 50 malignant gliomas that had received 1-(4-amino-2-methyl-5-pyrimidynyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after the resection of the tumor by real-time reverse transcription-polymerase chain reaction (RT-PCR) using TaqMan probe. 2. The mean absolute value of MGMTmRNA normalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for 50 tumors was 1.29 x 10(4)+/- 1.28 x 10(4) copy/microg RNA (mean +/- SD). The amount of MGMTmRNA less than 6 x 10(3) copy/microg RNA was the most significant factor in predicting the initial effect of treatment with ACNU by multi-variant regression analysis (p = 0.0157). 3. These results suggest that quantitation of MGMTmRNA is the excellent method for predicting for the effect of ACNU in glioma therapy.

Clinical Implications of Promoter Hypermethylation in RASSF1A and MGMT in Retinoblastoma1

PubMed Central

Choy, Kwong Wai; Lee, Tom C; Cheung, Kin Fai; Fan, Dorothy S P; Lo, Kwok Wai; Beaverson, Katherine L; Abramson, David H; Lam, Dennis S C; Yu, Christopher B O; Pang, Chi Pui

2005-01-01

Abstract We investigated the epigenetic silencing and genetic changes of the RAS-associated domain family 1A (RASSF1A) gene and the O6-methylguanine-DNA methyltransferase (MGMT) gene in retinoblastoma. We extracted DNA from microdissected tumor and normal retina tissues of the same patient in 68 retinoblastoma cases. Promoter methylation in RASSF1A and MGMT was analyzed by methylation-specific PCR, RASSF1A sequence alterations in all coding exons by direct DNA sequencing, and RASSF1A expression by RT-PCR. Cell cycle staging was analyzed by flow cytometry. We detected RASSF1A promoter hypermethylation in 82% of retinoblastoma, in tumor tissues only but not in adjacent normal retinal tissue cells. There was no expression of RASSF1A transcripts in all hypermethylated samples, but RASSF1A transcripts were restored after 5-aza-2′-deoxycytidine treatment with no changes in cell cycle or apoptosis. No mutation in the RASSF1A sequence was found. MGMT hypermethylation was present in 15% of theretinoblastoma samples, and the absence of MGMT hypermethylation was associated (P = .002) with retinoblastoma at advanced Reese-Ellsworth tumor stage. Our results revealed a high RASSF1A hypermethylation frequency in retinoblastoma. The correlation of MGMT inactivation by promoter hypermethylation with lower-stage diseases indicated that MGMT hypermethylation provides useful prognostic information. Epigenetic mechanism plays an important role in the progression of retinoblastoma. PMID:15799820

Mutagenic frequencies of site-specifically located O6-methylguanine in wild-type Escherichia coli and in a strain deficient in ada-methyltransferase.

PubMed

Rossi, S C; Topal, M D

1991-02-01

The adaptive response of Escherichia coli involves protection of the cells against the toxic and mutagenic consequences of exposure to high doses of a methylating agent by prior exposure to low doses of the agent. Ada protein, a major repair activity for O6-methylguanine, is activated to positively control the adaptive response; O6-methylguanine is one of the major mutagenic lesions produced by methylating agents. We investigated the mutation frequency of wild-type Escherichia coli and strains containing the ada-5 mutation in response to site-specifically synthesized O6-methylguanine under conditions in which the adaptive response was not induced. Site-directed mutagenesis and oligonucleotide self-selection techniques were used to isolate the progeny of M13mp18 DNAs constructed to contain O6-methylguanine at any of eight different positions. The progeny were isolated from E. coli strains isogeneic except for deficiency in Ada-methyltransferase repair, UvrABC excision repair, or both. The resulting O6-methylguanine mutation levels at each position were determined by using differential oligonucleotide hybridization. We found that the wild type had up to a 2.6-fold higher mutation frequency than ada-5 mutants. In addition, the mutation frequency varied with the position of the O6-methylguanine in the DNA in the wild type but not in ada-5 mutants; O6-methylguanine lesions at the 5' ends of runs of consecutive guanines gave the highest mutation frequencies. Determination of the mutation frequency of O6-methylguanine in wild-type and mutS cells showed that mismatch repair can affect O6-methylguanine mutation levels.

Correlation between MGMT promoter methylation and response to temozolomide-based therapy in neuroendocrine neoplasms: an observational retrospective multicenter study.

PubMed

Campana, Davide; Walter, Thomas; Pusceddu, Sara; Gelsomino, Fabio; Graillot, Emmanuelle; Prinzi, Natalie; Spallanzani, Andrea; Fiorentino, Michelangelo; Barritault, Marc; Dall'Olio, Filippo; Brighi, Nicole; Biasco, Guido

2018-06-01

Temozolomide (TEM) based therapy has been reported being effective in the treatment of metastatic neuroendocrine neoplasms (NEN), with response rates ranging from 30 to 70%. Among patients affected by advanced glioblastoma or melanoma and treated with TEM, loss of tumoral O6-methylguanine DNA methyltransferase (MGMT) is correlated with improved survival. In NEN patients, the role of MGMT deficiency in predicting clinical outcomes of TEM treatment is still under debate. In this study we evaluated 95 patients with advanced NENs undergoing treatment with TEM-based therapy. MGMT promoter methylation status was evaluated with two techniques: methylation specific-polymerase chain reaction or pyrosequencing. Treatment with TEM-based therapy was associated with an overall response rate of 27.4% according to RECIST criteria (51.8% of patients with and 17.7% without MGMT promoter methylation). Response to therapy, progression free survival and overall survival was correlated to MGMT status at univariate and multivariate analysis. Methylation of MGMT promoter could be a strong predictive factor of objective response and an important prognostic factor of a longer PFS and OS. According to our results, MGMT methylation status, evaluated with methylation specific-polymerase chain reaction or pyrosequencing, should have an important role in patients with metastatic NENs, in order to guide therapeutic options. These results need further confirmation with prospective studies.

Influence of promoter/enhancer region haplotypes on MGMT transcriptional regulation: a potential biomarker for human sensitivity to alkylating agents.

PubMed

Xu, Meixiang; Nekhayeva, Ilona; Cross, Courtney E; Rondelli, Catherine M; Wickliffe, Jeffrey K; Abdel-Rahman, Sherif Z

2014-03-01

The O6-methylguanine-DNA methyltransferase gene (MGMT) encodes the direct reversal DNA repair protein that removes alkyl adducts from the O6 position of guanine. Several single-nucleotide polymorphisms (SNPs) exist in the MGMT promoter/enhancer (P/E) region. However, the haplotype structure encompassing these SNPs and their functional/biological significance are currently unknown. We hypothesized that MGMT P/E haplotypes, rather than individual SNPs, alter MGMT transcription and can thus alter human sensitivity to alkylating agents. To identify the haplotype structure encompassing the MGMT P/E region SNPs, we sequenced 104 DNA samples from healthy individuals and inferred the haplotypes using the data generated. We identified eight SNPs in this region, namely T7C (rs180989103), T135G (rs1711646), G290A (rs61859810), C485A (rs1625649), C575A (rs113813075), G666A (rs34180180), C777A (rs34138162) and C1099T (rs16906252). Phylogenetics and Sequence Evolution analysis predicted 21 potential haplotypes that encompass these SNPs ranging in frequencies from 0.000048 to 0.39. Of these, 10 were identified in our study population as 20 paired haplotype combinations. To determine the functional significance of these haplotypes, luciferase reporter constructs representing these haplotypes were transfected into glioblastoma cells and their effect on MGMT promoter activity was determined. Compared with the most common (reference) haplotype 1, seven haplotypes significantly upregulated MGMT promoter activity (18-119% increase; P < 0.05), six significantly downregulated MGMT promoter activity (29-97% decrease; P < 0.05) and one haplotype had no effect. Mechanistic studies conducted support the conclusion that MGMT P/E haplotypes, rather than individual SNPs, differentially regulate MGMT transcription and could thus play a significant role in human sensitivity to environmental and therapeutic alkylating agents.

EG-15THE METHYLATION STATUS OF MGMT IN MEDULLOBLASTOMA

PubMed Central

Shimizu, Yuzaburo; Kurimoto, Tomoko; Kondo, Akihide; Arai, Hajime

2014-01-01

BACKGROUND: Medulloblastoma is a highly malignant brain tumor in childhood. Some studies reported that alkylating chemotherapeutic drugs are effective agents in the treatment of patients with medulloblastoma. O6-methylguanine-DNA methyltransferase (MGMT) is one of the DNA repair enzymes and plays a significant role in tumor resistance to alkylating agents. Low MGMT expression or MGMT promoter methylation have been found to be associated with favorable outcomes in malignant glioma patients treated with alkylating agents such as temozolomide. However, impact of MGMT status on clinical outcomes in medulloblastoma patients is not fully evaluated. OBJECTIVE: The objective of this study is to investigate the association between MGMT status and the response for chemotherapy in pediatric patients with medulloblastoma. METHODS: Patients with medulloblastoma treated at our institution between 1995 and 2012 were reviewed retrospectively. Relevant clinical information including current disease status, tumor response to chemotherapy was obtained from the hospital charts. To evaluate the MGMT status, we performed bisulfite sequencing analysis to determine the methylation status of the MGMT promoter. RESULTS: Tumor material and detailed clinical information were available in 22 patients. Of them, 13 patients were alive (11 in CR), seven died of disease and two were lost to follow up. Five patients were with dissemination at diagnosis. We succeeded to evaluate both the MGMT status of tumors and the number of methylation sites in MGMT promoter. CONCLUSIONS: We studied the prognostic value of MGMT promoter methylation in medulloblastoma children.

Inhibition of Y-box binding protein-1 slows the growth of glioblastoma multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA methyltransferase.

PubMed

Gao, Yuanyuan; Fotovati, Abbas; Lee, Cathy; Wang, Michelle; Cote, Gilbert; Guns, Emma; Toyota, Brian; Faury, Damien; Jabado, Nada; Dunn, Sandra E

2009-12-01

Glioblastoma multiforme (GBM) is an aggressive type of brain tumor where <3% of newly diagnosed cases in the patients will survive >5 years. In adults, GBM is the most common type of brain tumor. It is rarer in children, where it constitutes approximately 15% of all brain tumors diagnosed. These tumors are often invasive, making surgical resection difficult. Further, they can be refractory to current therapies such as temozolomide. The current dogma is that temozolomide resistance rests on the expression of O6-methylguanine-DNA methyltransferase (MGMT) because it cleaves methylated DNA adducts formed by the drug. Our laboratory recently reported that another drug resistance gene known as the Y-box binding protein-1 (YB-1) is highly expressed in primary GBM but not in normal brain tissues based on the evaluation of primary tumors. We therefore questioned whether GBM depend on YB-1 for growth and/or response to temozolomide. Herein, we report that YB-1 inhibition reduced tumor cell invasion and growth in monolayer as well as in soft agar. Moreover, blocking this protein ultimately delayed tumor onset in mice. Importantly, inhibiting YB-1 enhanced temozolomide sensitivity in a manner that was independent of MGMT in models of adult and pediatric GBM. In conclusion, inhibiting YB-1 may be a novel way to improve the treatment of GBM.

Treatment of recurrent malignant gliomas with fotemustine monotherapy: impact of dose and correlation with MGMT promoter methylation.

PubMed

Fabi, Alessandra; Metro, Giulio; Russillo, Michelangelo; Vidiri, Antonello; Carapella, Carmine Maria; Maschio, Marta; Cognetti, Francesco; Jandolo, Bruno; Mirri, Maria Alessandra; Sperduti, Isabella; Telera, Stefano; Carosi, Mariantonia; Pace, Andrea

2009-03-31

In recurrent malignant gliomas (MGs), a high rate of haematological toxicity is observed with the use of fotemustine at the conventional schedule (100 mg/m(2) weekly for 3 consecutive weeks followed by triweekly administration after a 5-week rest period). Also, the impact of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status on fotemustine activity has never been explored in the clinical setting. 40 patients with recurrent pretreated MG were identified as being treated with fotemustine at doses ranging from 65 mg/m(2) to 100 mg/m(2). Patients were classified into 3 groups according to the dose of fotemustine received, from the lowest dosage received in group A, to the highest in group C. Analysis of MGMT promoter methylation in tumor tissue was successfully performed in 19 patients. Overall, 20% of patients responded to treatment, for a disease control rate (DCR, responses plus stabilizations) of 47.5%. Groups A and B experienced a response rate of 40% and 26.5% respectively, while the corresponding value for group C was 10%. Out of 19 patients, MGMT promoter was found methylated in 12 cases among which a DCR of 66.5% was observed. All 7 patients with unmethylated MGMT promoter were progressive to fotemustine. Low-dose fotemustine at 65-75 mg/m(2) (induction phase) followed by 75-85 mg/m(2) (maintenance phase) has an activity comparable to that of the conventional schedule. By determination of the MGMT promoter methylation status patients might be identified who are more likely to benefit from fotemustine chemotherapy.

Promoter CpG island hypermethylation of the DNA repair enzyme MGMT predicts clinical response to dacarbazine in a phase II study for metastatic colorectal cancer.

PubMed

Amatu, Alessio; Sartore-Bianchi, Andrea; Moutinho, Catia; Belotti, Alessandro; Bencardino, Katia; Chirico, Giuseppe; Cassingena, Andrea; Rusconi, Francesca; Esposito, Anna; Nichelatti, Michele; Esteller, Manel; Siena, Salvatore

2013-04-15

O(6)-methylguanine-DNA-methyltransferase (MGMT) is a DNA repair protein removing mutagenic and cytotoxic adducts from O(6)-guanine in DNA. Approximately 40% of colorectal cancers (CRC) display MGMT deficiency due to the promoter hypermethylation leading to silencing of the gene. Alkylating agents, such as dacarbazine, exert their antitumor activity by DNA methylation at the O(6)-guanine site, inducing base pair mismatch; therefore, activity of dacarbazine could be enhanced in CRCs lacking MGMT. We conducted a phase II study with dacarbazine in CRCs who had failed standard therapies (oxaliplatin, irinotecan, fluoropyrimidines, and cetuximab or panitumumab if KRAS wild-type). All patients had tumor tissue assessed for MGMT as promoter hypermethylation in double-blind for treatment outcome. Patients received dacarbazine 250 mg/m(2) intravenously every day for four consecutive days, every 21 days, until progressive disease or intolerable toxicity. We used a Simon two-stage design to determine whether the overall response rate would be 10% or more. Secondary endpoints included association of response, progression-free survival, and disease control rate with MGMT status. Sixty-eight patients were enrolled from May 2011 to March 2012. Patients received a median of three cycles of dacarbazine (range 1-12). Grades 3 and 4 toxicities included: fatigue (41%), nausea/vomiting (29%), constipation (25%), platelet count decrease (19%), and anemia (18%). Overall, two patients (3%) achieved partial response and eight patients (12%) had stable disease. Disease control rate (partial response + stable disease) was significantly associated with MGMT promoter hypermethylation in the corresponding tumors. Objective clinical responses to dacarbazine in patients with metastatic CRC are confined to those tumors harboring epigenetic inactivation of the DNA repair enzyme MGMT.

Enhanced O6-methylguanine-DNA methyltransferase activity in transgenic mice containing an integrated E. coli ada repair gene.

PubMed

Matsukuma, S; Nakatsuru, Y; Nakagawa, K; Utakoji, T; Sugano, H; Kataoka, H; Sekiguchi, M; Ishikawa, T

1989-11-01

The E. coli ada gene encodes O6-methylguanine DNA methyltransferase (O6MTase) which repairs the methylation of guanine at the O6 position in DNA. After recombination with a Chinese hamster metallothionein I gene promoter, the ada gene was microinjected into C3H/HeN mouse zygotes. Eventually, transgenic mice containing the ada fusion DNA were generated. The integrated ada DNA complex was transmitted to the progeny in a mode conforming to tandem integration at a single chromosome site, and homozygotes were also obtained from an inter-transgenic mouse cross. RNA transcripts of the chimeric ada gene were identified in the livers of these transgenic mice using dot and Northern blot analyses. O6MTase activity was increased in the liver of transgenic mice of line No. 708, and was more than 3 times the activity found in non-transgenic mice, especially in the transgenic homozygotes. The ada gene product was detected in the liver of a transgenic homozygote by immunoblot analysis. These transgenic mice have great potential for analysis of the role played by O6MTase in chemical carcinogenesis.

MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response.

PubMed

Switzeny, Olivier J; Christmann, Markus; Renovanz, Mirjam; Giese, Alf; Sommer, Clemens; Kaina, Bernd

2016-01-01

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value. We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP. Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

Using the apparent diffusion coefficient to identifying MGMT promoter methylation status early in glioblastoma: importance of analytical method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rundle-Thiele, Dayle; Day, Bryan; Stringer, Brett

Accurate knowledge of O{sup 6}-methylguanine methyltransferase (MGMT) gene promoter subtype in patients with glioblastoma (GBM) is important for treatment. However, this test is not always available. Pre-operative diffusion MRI (dMRI) can be used to probe tumour biology using the apparent diffusion coefficient (ADC); however, its ability to act as a surrogate to predict MGMT status has shown mixed results. We investigated whether this was due to variations in the method used to analyse ADC. We undertook a retrospective study of 32 patients with GBM who had MGMT status measured. Matching pre-operative MRI data were used to calculate the ADC withinmore » contrast enhancing regions of tumour. The relationship between ADC and MGMT was examined using two published ADC methods. A strong trend between a measure of ‘minimum ADC’ and methylation status was seen. An elevated minimum ADC was more likely in the methylated compared to the unmethylated MGMT group (U = 56, P = 0.0561). In contrast, utilising a two-mixture model histogram approach, a significant reduction in mean measure of the ‘low ADC’ component within the histogram was associated with an MGMT promoter methylation subtype (P < 0.0246). This study shows that within the same patient cohort, the method selected to analyse ADC measures has a significant bearing on the use of that metric as a surrogate marker of MGMT status. Thus for dMRI data to be clinically useful, consistent methods of data analysis need to be established prior to establishing any relationship with genetic or epigenetic profiling.« less

MGMT promoter methylation in Peruvian patients with glioblastoma

PubMed Central

Belmar-Lopez, Carolina; Castaneda, Carlos A; Castillo, Miluska; García-Corrochano, Pamela; Orrego, Enrique; Meléndez, Barbara; Casavilca, Sandro; Flores, Claudio; Orrego, Enrique

2018-01-01

Purpose O6-methylguanine–DNA methyltransferase (MGMT) promoter methylation predicts the outcome and response to alkylating chemotherapy in glioblastoma. The aim of this study is to evaluate the prevalence of MGMT methylation in Peruvian glioblastoma cases. Patients and methods We evaluated retrospectively 50 cases of resected glioblastoma during the period 2008–2013 at Instituto Nacional de Enfermedades Neoplasicas in Peru. Samples consisted of paraffin embedded and frozen tumour tissue. MGMT-promoter methylation status and the expression level of MGMT gene were evaluated by methylation-specific PCR and real-time PCR, respectively. Results Unmethylated, methylated and partially methylated statuses were found in 54%, 20% and 26% of paraffin-embedded samples, respectively. Methylation status was confirmed in the Virgen de la Salud Hospital and frozen samples. There was an association between the status of MGMT-promoter methylation and the level of gene expression (p = 0.001). Methylation was associated with increased progression-free survival (p = 0.002) and overall survival (OS) (p < 0.001). Conclusion MGMT-promoter methylation frequency in Peruvian glioblastoma is similar to that reported in other populations and the detection test has been standardised. PMID:29515653

Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas

PubMed Central

Everhard, Sibille; Tost, Jörg; Abdalaoui, Hafida El; Crinière, Emmanuelle; Busato, Florence; Marie, Yannick; Gut, Ivo G.; Sanson, Marc; Mokhtari, Karima; Laigle-Donadey, Florence; Hoang-Xuan, Khê; Delattre, Jean-Yves; Thillet, Joëlle

2009-01-01

The O6-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sites that are potentially related to expression was investigated in 54 glioblastomas by pyrosequencing, a highly quantitative method, and analyzed with respect to their MGMT mRNA expression status. Three groups of patients were identified according to the methylation pattern of all 52 analyzed CpG sites. Overall, an 85% rate of concordance was observed between methylation and expression (p < 0.0001). When analyzing each CpG separately, six CpG sites were highly correlated with expression (p < 0.0001), and two CpG regions could be used as surrogate markers for RNA expression in 81.5% of the patients. This study indicates that there is good statistical agreement between MGMT methylation and expression, and that some CpG regions better reflect MGMT expression than do others. However, if transcriptional repression is the key mechanism in explaining the higher chemosensitivity of MGMT-methylated tumors, a substantial rate of discordance should lead clinicians to be cautious when deciding on a therapeutic strategy based on MGMT methylation status alone. PMID:19224763

Identification of regions correlating MGMT promoter methylation and gene expression in glioblastomas.

PubMed

Everhard, Sibille; Tost, Jörg; El Abdalaoui, Hafida; Crinière, Emmanuelle; Busato, Florence; Marie, Yannick; Gut, Ivo G; Sanson, Marc; Mokhtari, Karima; Laigle-Donadey, Florence; Hoang-Xuan, Khê; Delattre, Jean-Yves; Thillet, Joëlle

2009-08-01

The O(6)-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sites that are potentially related to expression was investigated in 54 glioblastomas by pyrosequencing, a highly quantitative method, and analyzed with respect to their MGMT mRNA expression status. Three groups of patients were identified according to the methylation pattern of all 52 analyzed CpG sites. Overall, an 85% rate of concordance was observed between methylation and expression (p < 0.0001). When analyzing each CpG separately, six CpG sites were highly correlated with expression (p < 0.0001), and two CpG regions could be used as surrogate markers for RNA expression in 81.5% of the patients. This study indicates that there is good statistical agreement between MGMT methylation and expression, and that some CpG regions better reflect MGMT expression than do others. However, if transcriptional repression is the key mechanism in explaining the higher chemosensitivity of MGMT-methylated tumors, a substantial rate of discordance should lead clinicians to be cautious when deciding on a therapeutic strategy based on MGMT methylation status alone.

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

PubMed

Moore, M H; Gulbis, J M; Dodson, E J; Demple, B; Moody, P C

1994-04-01

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

DNA methylation and histone acetylation regulate the expression of MGMT and chemosensitivity to temozolomide in malignant melanoma cell lines.

PubMed

Chen, Ya-Ping; Hou, Xiao-Yang; Yang, Chun-Sheng; Jiang, Xiao-Xiao; Yang, Ming; Xu, Xi-Feng; Feng, Shou-Xin; Liu, Yan-Qun; Jiang, Guan

2016-08-01

Malignant melanoma is an aggressive, highly lethal dermatological malignancy. Chemoresistance and rapid metastasis limit the curative effect of multimodal therapies like surgery or chemotherapy. The suicide enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes adducts from the O6-position of guanine to repair DNA damage. High MGMT expression is associated with resistance to therapy in melanoma. However, it is unknown if MGMT is regulated by DNA methylation or histone acetylation in melanoma. We examined the effects of the DNA methylation inhibitor 5-Aza-2'-deoxycytidine and histone deacetylase inhibitor Trichostatin A alone or in combination on MGMT expression and promoter methylation and histone acetylation in A375, MV3, and M14 melanoma cells. This study demonstrates that MGMT expression, CpG island methylation, and histone acetylation vary between melanoma cell lines. Combined treatment with 5-Aza-2'-deoxycytidine and Trichostatin A led to reexpression of MGMT, indicating that DNA methylation and histone deacetylation are associated with silencing of MGMT in melanoma. This study provides information on the role of epigenetic modifications in malignant melanoma that may enable the development of new strategies for treating malignant melanoma.

Comprehensive analysis of MGMT promoter methylation: correlation with MGMT expression and clinical response in GBM.

PubMed

Shah, Nameeta; Lin, Biaoyang; Sibenaller, Zita; Ryken, Timothy; Lee, Hwahyung; Yoon, Jae-Geun; Rostad, Steven; Foltz, Greg

2011-01-07

O⁶-methylguanine DNA-methyltransferase (MGMT) promoter methylation has been identified as a potential prognostic marker for glioblastoma patients. The relationship between the exact site of promoter methylation and its effect on gene silencing, and the patient's subsequent response to therapy, is still being defined. The aim of this study was to comprehensively characterize cytosine-guanine (CpG) dinucleotide methylation across the entire MGMT promoter and to correlate individual CpG site methylation patterns to mRNA expression, protein expression, and progression-free survival. To best identify the specific MGMT promoter region most predictive of gene silencing and response to therapy, we determined the methylation status of all 97 CpG sites in the MGMT promoter in tumor samples from 70 GBM patients using quantitative bisulfite sequencing. We next identified the CpG site specific and regional methylation patterns most predictive of gene silencing and improved progression-free survival. Using this data, we propose a new classification scheme utilizing methylation data from across the entire promoter and show that an analysis based on this approach, which we call 3R classification, is predictive of progression-free survival (HR  = 5.23, 95% CI [2.089-13.097], p<0.0001). To adapt this approach to the clinical setting, we used a methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) test based on the 3R classification and show that this test is both feasible in the clinical setting and predictive of progression free survival (HR  = 3.076, 95% CI [1.301-7.27], p = 0.007). We discuss the potential advantages of a test based on this promoter-wide analysis and compare it to the commonly used methylation-specific PCR test. Further prospective validation of these two methods in a large independent patient cohort will be needed to confirm the added value of promoter wide analysis of MGMT methylation in the clinical setting.

MGMT inactivation and clinical response in newly diagnosed GBM patients treated with Gliadel.

PubMed

Grossman, Rachel; Burger, Peter; Soudry, Ethan; Tyler, Betty; Chaichana, Kaisorn L; Weingart, Jon; Olivi, Alessandro; Gallia, Gary L; Sidransky, David; Quiñones-Hinojosa, Alfredo; Ye, Xiaobu; Brem, Henry

2015-12-01

We examined the relationship between the O(6)-methylguanine-methyltransferase (MGMT) methylation status and clinical outcomes in newly diagnosed glioblastoma multiforme (GBM) patients who were treated with Gliadel wafers (Eisai, Tokyo, Japan). MGMT promoter methylation has been associated with increased survival among patients with GBM who are treated with various alkylating agents. MGMT promoter methylation, in DNA from 122 of 160 newly diagnosed GBM patients treated with Gliadel, was determined by a quantitative methylation-specific polymerase chain reaction, and was correlated with overall survival (OS) and recurrence-free survival (RFS). The MGMT promoter was methylated in 40 (32.7%) of 122 patients. The median OS was 13.5 months (95% confidence interval [CI] 11.0-14.5) and RFS was 9.4 months (95% CI 7.8-10.2). After adjusting for age, Karnofsky performance score, extent of resection, temozolomide (TMZ) and radiation therapy (RT), the newly diagnosed GBM patients with MGMT methylation had a 15% reduced mortality risk, compared to patients with unmethylated MGMT (hazard ratio 0.85; 95% CI 0.56-1.31; p=0.46). The patients aged over 70 years with MGMT methylation had a significantly longer median OS of 13.5 months, compared to 7.6 months in patients with unmethylated MGMT (p=0.027). A significant difference was also found in older patients, with a median RFS of 13.1 versus 7.6 months for methylated and unmethylated MGMT groups, respectively (p=0.01). Methylation of the MGMT promoter in newly diagnosed GBM patients treated with Gliadel, RT and TMZ, was associated with significantly improved OS compared to the unmethylated population. In elderly patients, methylation of the MGMT promoter was associated with significantly better OS and RFS. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human TERT promoter mutation enables survival advantage from MGMT promoter methylation in IDH1 wild-type primary glioblastoma treated by standard chemoradiotherapy.

PubMed

Nguyen, HuyTram N; Lie, Amy; Li, Tie; Chowdhury, Reshmi; Liu, Fei; Ozer, Byram; Wei, Bowen; Green, Richard M; Ellingson, Benjamin M; Wang, He-Jing; Elashoff, Robert; Liau, Linda M; Yong, William H; Nghiemphu, Phioanh L; Cloughesy, Timothy; Lai, Albert

2017-03-01

Promoter mutation in the human telomerase reverse transcriptase gene (hTERT) occurs in ~75% of primary glioblastoma (GBM). Although the mutation appears to upregulate telomerase expression and contributes to the maintenance of telomere length, its clinical significance remains unclear. We performed hTERT promoter genotyping on 303 isocitrate dehydrogenase 1 wild-type GBM tumors treated with standard chemoradiotherapy. We also stratified 190 GBM patients from the database of The Cancer Genome Atlas (TCGA) by hTERT gene expression. We analyzed overall and progression-free survival by Kaplan-Meier and Cox regression. We detected hTERT promoter mutation in 75% of the patients. When included as the only biomarker, hTERT mutation was not prognostic in our patient cohort by Cox regression analysis. However, when hTERT and O6-DNA methylguanine-methyltransferase (MGMT) were included together, we observed an interaction between these 2 factors. To further investigate this interaction, we performed pairwise comparison of the 4 patient subcohorts grouped by hTERT-MGMT status (MUT-M, WT-M, MUT-U, and WT-U). MGMT methylated patients showed improved survival only in the presence of hTERT promoter mutation: MUT-M versus MUT-U (overall survival of 28.3 vs 15.9 mos, log-rank P < .0001 and progression-free survival of 15.4 vs 7.86 mo, log-rank P < .0001). These results were confirmed by Cox analyses. Analogously, the cohort from TCGA demonstrated survival benefit of MGMT promoter methylation only in patients with high hTERT expression. In addition, hTERT mutation was negatively prognostic in our MGMT unmethylated patients, while the analogous association with high expression was not observed in the cohort from TCGA. The prognostic influence of MGMT promoter methylation depends on hTERT promoter mutation. This interaction warrants further mechanistic investigation. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights

Molecular correlates with MGMT promoter methylation and silencing support CpG island methylator phenotype-low (CIMP-low) in colorectal cancer.

PubMed

Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Suemoto, Yuko; Meyerhardt, Jeffrey A; Fuchs, Charles S

2007-11-01

The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer. In contrast, a phenotype with less widespread promoter methylation (CIMP-low) has not been well characterised. O-6-methylguanine-DNA methyltransferase (MGMT) promoter methylation and silencing have been associated with G>A mutations and microsatellite instability-low (MSI-low). To examine molecular correlates with MGMT methylation/silencing in colorectal cancer. Utilising MethyLight technology, we quantified DNA methylation in MGMT and eight other markers (a CIMP-diagnostic panel; CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1) in 920 population-based colorectal cancers. Tumours with both MGMT methylation and loss were correlated positively with MSI-low (p = 0.02), CIMP-high (>or=6/8 methylated CIMP markers, p = 0.005), CIMP-low (1/8-5/8 methylated CIMP markers, p = 0.002, compared to CIMP-0 with 0/8 methylated markers), KRAS G>A mutation (p = 0.02), and inversely with 18q loss of heterozygosity (p = 0.0002). Tumours were classified into nine MSI/CIMP subtypes. Among the CIMP-low group, tumours with both MGMT methylation and loss were far more frequent in MSI-low tumours (67%, 12/18) than MSI-high tumours (5.6%, 1/18; p = 0.0003) and microsatellite stable (MSS) tumours (33%, 52/160; p = 0.008). However, no such relationship was observed among the CIMP-high or CIMP-0 groups. The relationship between MGMT methylation/silencing and MSI-low is limited to only CIMP-low tumours, supporting the suggestion that CIMP-low in colorectal cancer may be a different molecular phenotype from CIMP-high and CIMP-0. Our data support a molecular difference between MSI-low and MSS in colorectal cancer, and a possible link between CIMP-low, MSI-low, MGMT methylation/loss and KRAS mutation.

Variation in the loss of O6-methylguanine-DNA methyltransferase during immortalization of human fibroblasts.

PubMed

Green, M H; Karran, P; Lowe, J E; Priestley, A; Arlett, C F; Mayne, L

1990-01-01

We have examined O6-methylguanine-DNA methyltransferase (MT) activity in four human fibroblast cell lines during immortalization. Transfection of primary fibroblasts with the plasmid pSV3gpt or pSV3neo, which encode the SV40 large T antigen, confers a transformed phenotype but not immediate immortality. After a period of growth (pre-crisis) the cells enter a quiescent phase (crisis) from which an immortal clone of cells eventually grows out. From measurements of MT activity in extracts of cells taken at different defined stages of the immortalization process, we conclude that the establishment of a Mex- (MT-deficient) cell population is not specifically associated with cellular transformation or with any particular stage of immortalization. It appears that in different cell populations the change from Mex+ to Mex- may occur at different times during the immortalization process and that the change may be very abrupt.

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy.

PubMed

Weiler, Markus; Blaes, Jonas; Pusch, Stefan; Sahm, Felix; Czabanka, Marcus; Luger, Sebastian; Bunse, Lukas; Solecki, Gergely; Eichwald, Viktoria; Jugold, Manfred; Hodecker, Sibylle; Osswald, Matthias; Meisner, Christoph; Hielscher, Thomas; Rübmann, Petra; Pfenning, Philipp-Niklas; Ronellenfitsch, Michael; Kempf, Tore; Schnölzer, Martina; Abdollahi, Amir; Lang, Florian; Bendszus, Martin; von Deimling, Andreas; Winkler, Frank; Weller, Michael; Vajkoczy, Peter; Platten, Michael; Wick, Wolfgang

2014-01-07

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O(6)-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids.

mTOR target NDRG1 confers MGMT-dependent resistance to alkylating chemotherapy

PubMed Central

Weiler, Markus; Blaes, Jonas; Pusch, Stefan; Sahm, Felix; Czabanka, Marcus; Luger, Sebastian; Bunse, Lukas; Solecki, Gergely; Eichwald, Viktoria; Jugold, Manfred; Hodecker, Sibylle; Osswald, Matthias; Meisner, Christoph; Hielscher, Thomas; Rübmann, Petra; Pfenning, Philipp-Niklas; Ronellenfitsch, Michael; Kempf, Tore; Schnölzer, Martina; Abdollahi, Amir; Lang, Florian; Bendszus, Martin; von Deimling, Andreas; Winkler, Frank; Weller, Michael; Vajkoczy, Peter; Platten, Michael; Wick, Wolfgang

2014-01-01

A hypoxic microenvironment induces resistance to alkylating agents by activating targets in the mammalian target of rapamycin (mTOR) pathway. The molecular mechanisms involved in this mTOR-mediated hypoxia-induced chemoresistance, however, are unclear. Here we identify the mTOR target N-myc downstream regulated gene 1 (NDRG1) as a key determinant of resistance toward alkylating chemotherapy, driven by hypoxia but also by therapeutic measures such as irradiation, corticosteroids, and chronic exposure to alkylating agents via distinct molecular routes involving hypoxia-inducible factor (HIF)-1alpha, p53, and the mTOR complex 2 (mTORC2)/serum glucocorticoid-induced protein kinase 1 (SGK1) pathway. Resistance toward alkylating chemotherapy but not radiotherapy was dependent on NDRG1 expression and activity. In posttreatment tumor tissue of patients with malignant gliomas, NDRG1 was induced and predictive of poor response to alkylating chemotherapy. On a molecular level, NDRG1 bound and stabilized methyltransferases, chiefly O6-methylguanine-DNA methyltransferase (MGMT), a key enzyme for resistance to alkylating agents in glioblastoma patients. In patients with glioblastoma, MGMT promoter methylation in tumor tissue was not more predictive for response to alkylating chemotherapy in patients who received concomitant corticosteroids. PMID:24367102

MRI texture features as biomarkers to predict MGMT methylation status in glioblastomas

DOE Office of Scientific and Technical Information (OSTI.GOV)

Korfiatis, Panagiotis; Kline, Timothy L.; Erickson, Bradley J., E-mail: bje@mayo.edu

Purpose: Imaging biomarker research focuses on discovering relationships between radiological features and histological findings. In glioblastoma patients, methylation of the O{sup 6}-methylguanine methyltransferase (MGMT) gene promoter is positively correlated with an increased effectiveness of current standard of care. In this paper, the authors investigate texture features as potential imaging biomarkers for capturing the MGMT methylation status of glioblastoma multiforme (GBM) tumors when combined with supervised classification schemes. Methods: A retrospective study of 155 GBM patients with known MGMT methylation status was conducted. Co-occurrence and run length texture features were calculated, and both support vector machines (SVMs) and random forest classifiersmore » were used to predict MGMT methylation status. Results: The best classification system (an SVM-based classifier) had a maximum area under the receiver-operating characteristic (ROC) curve of 0.85 (95% CI: 0.78–0.91) using four texture features (correlation, energy, entropy, and local intensity) originating from the T2-weighted images, yielding at the optimal threshold of the ROC curve, a sensitivity of 0.803 and a specificity of 0.813. Conclusions: Results show that supervised machine learning of MRI texture features can predict MGMT methylation status in preoperative GBM tumors, thus providing a new noninvasive imaging biomarker.« less

Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

PubMed Central

Fukai, Junya; Koizumi, Fumiaki; Nakao, Naoyuki

2014-01-01

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O6-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance. PMID:25111384

Efficacy of protracted temozolomide dosing is limited in MGMT unmethylated GBM xenograft models.

PubMed

Cen, Ling; Carlson, Brett L; Pokorny, Jenny L; Mladek, Ann C; Grogan, Patrick T; Schroeder, Mark A; Decker, Paul A; Anderson, S Keith; Giannini, Caterina; Wu, Wenting; Ballman, Karla V; Kitange, Gaspar J; Sarkaria, Jann N

2013-06-01

Temozolomide (TMZ) is important chemotherapy for glioblastoma multiforme (GBM), but the optimal dosing schedule is unclear. The efficacies of different clinically relevant dosing regimens were compared in a panel of 7 primary GBM xenografts in an intracranial therapy evaluation model. Protracted TMZ therapy (TMZ daily M-F, 3 wk every 4) provided superior survival to a placebo-treated group in 1 of 4 O(6)-DNA methylguanine-methyltransferase (MGMT) promoter hypermethylated lines (GBM12) and none of the 3 MGMT unmethylated lines, while standard therapy (TMZ daily M-F, 1 wk every 4) provided superior survival to the placebo-treated group in 2 of 3 MGMT unmethylated lines (GBM14 and GBM43) and none of the methylated lines. In comparing GBM12, GBM14, and GBM43 intracranial specimens, both GBM14 and GBM43 mice treated with protracted TMZ had a significant elevation in MGMT levels compared with placebo. Similarly, high MGMT was found in a second model of acquired TMZ resistance in GBM14 flank xenografts, and resistance was reversed in vitro by treatment with the MGMT inhibitor O(6)-benzylguanine, demonstrating a mechanistic link between MGMT overexpression and TMZ resistance in this line. Additionally, in an analysis of gene expression data, comparison of parental and TMZ-resistant GBM14 demonstrated enrichment of functional ontologies for cell cycle control within the S, G2, and M phases of the cell cycle and DNA damage checkpoints. Across the 7 tumor models studied, there was no consistent difference between protracted and standard TMZ regimens. The efficacy of protracted TMZ regimens may be limited in a subset of MGMT unmethylated tumors by induction of MGMT expression.

Research on DNA methylation of human osteosarcoma cell MGMT and its relationship with cell resistance to alkylating agents.

PubMed

Guo, Jun; Cui, Qiu; Jiang, WeiHao; Liu, Cheng; Li, DingFeng; Zeng, Yanjun

2013-08-01

The objective of this study was to explore the O(6)-methylguanine-DNA methyltransferase (MGMT) gene methylation status and its protein expression, as well as the effects of demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-CdR) on MGMT gene expression and its resistance to alkylating agents, and to elucidate MGMT expression mechanism and significance in osteosarcoma. The human osteosarcoma cell lines Saos-2 and MG-63 were collected and treated with 5-Aza-CdR for 6 days. The cells not treated with 5-Aza-CdR were set as a negative control. The genomic DNA was extracted from the Saos-2 and MG-63 cells using methylation-specific PCR to detect the promoter CpG island methylation status of the MGMT gene. Cell sensitivity to alkylating agents before and after drug administration was detected by the MTT method. The variation in MGMT gene mRNA and protein was detected by reverse transcription PCR (RT-PCR) and Western blotting. The MGMT promoter gene of normal Saos-2 cells was methylated, with reduced MGMT mRNA and protein expression; the MGMT mRNA and protein expression of Saos-2 cells treated with 5-Aza-CdR was obviously enhanced, and its sensitivity to alkylating agents was reversed. Meanwhile, with promoter CpG island unmethylation of the MGMT gene, MGMT protein was expressed in the normal MG-63 cells and the MG-63 cells treated with 5-Aza-CdR, and both showed resistance to alkylating agents. The methylation status of the MGMT gene promoter in human osteosarcoma cells reflected the cells' ability to induce MGMT protein expression and can be used as a molecular marker to project the sensitivity of cancer tissues to alkylating agent drugs.

Concordant association validates MGMT methylation and protein expression as favorable prognostic factors in glioma patients on alkylating chemotherapy (Temozolomide).

PubMed

Pandith, Arshad A; Qasim, Iqbal; Zahoor, Wani; Shah, Parveen; Bhat, Abdul R; Sanadhya, Dheera; Shah, Zafar A; Naikoo, Niyaz A

2018-04-30

O 6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation and its subsequent loss of protein expression has been identified to have a variable impact on clinical outcome of glioma patients indicated for chemotherapy with alkylating agents (Temozolomide). This study investigated methylation status of MGMT gene along with in situ protein expression in malignant glioma patients of different histological types to evaluate the associated clinical outcome vis-a-vis use of alkylating drugs and radiotherapy. Sixty three cases of glioma were evaluated for MGMT promoter methylation by methylation-specific PCR (MS-PCR) and protein expression by immunostaining (IHC). Methylation status of MGMT and loss of protein expression showed a very high concordant association with better survival and progression free survival (PFS) (p < 0.0001). Multivariate Cox regression analysis showed both MGMT methylation and loss of protein as significant independent prognostic factors in glioma patients with respect to lower Hazard Ratio (HR) for better OS and PFS) [p < 0.05]. Interestingly concordant MGMT methylation and lack of protein showed better response in TMZ therapy treated patient subgroups with HR of 2.02 and 0.76 (p < 0.05). We found the merits of prognostication of MGMT parameters, methylation as well as loss of its protein as predictive factors for favorable outcome in terms of better survival for TMZ therapy.

Overcoming Temozolomide Resistance in Glioblastoma Multiforme with MGMT-Targeting Spherical Nucleic Acids

NASA Astrophysics Data System (ADS)

Sita, Timothy L.

Glioblastoma multiforme (GBM) is the most prevalent primary central nervous system malignancy. Due to the aggressive nature of these tumors and our inability to adequately treat them, only 3-5% of patients survive longer than 3 years post-diagnosis. The standard of care for newly diagnosed GBM is surgical resection followed by concomitant and adjuvant radiotherapy and temozolomide (TMZ) chemotherapy. TMZ cytotoxicity is mediated primarily through methylation of the O 6 -position of guanine. In the majority of patients, this methyl group is rapidly removed by the enzyme O6 -methylguanine-DNA methyltransferase (MGMT), conferring resistance to the chemotherapy. However, in a small subset of GBM patients, the promoter region for MGMT is methylated over the course of tumor development. This epigenetic silencing of MGMT activity allows TMZ to induce apoptosis in glioblastoma cells and drastically increases survival in GBM patients. The following work seeks to recapitulate this improved survival phenotype by combining TMZ with a novel nanoconstruct capable of silencing MGMT expression. The nanoconstruct consists of gold nanoparticles densely conjugated with either an MGMT-targeting ribozyme (ribozyme-Spherical Nucleic Acids (SNAs)), or small interfering RNA (siRNA) duplexes designed against MGMT (siMGMT-SNAs), and has been found to have unique characteristics, including (1) the rapid internalization by all glioma cell types studied including glioma initiating cells (GICs), (2) the capacity to potently silence MGMT expression, (3) increased apoptotic response in GBM cells, (4) the ability to cross the blood-brain barrier (BBB), blood-tumor barrier (BTB), and accumulate in GBM xenografts, and (5) no observable acute toxicity at high doses in animal models. In summary, preliminary data suggest ribozyme-SNA and siMGMT-SNAs sensitize GBM cells in vitro and in vivo, enhancing the therapeutic response to TMZ.

Assessing CpG island methylator phenotype, 1p/19q codeletion, and MGMT promoter methylation from epigenome-wide data in the biomarker cohort of the NOA-04 trial

PubMed Central

Wiestler, Benedikt; Capper, David; Hovestadt, Volker; Sill, Martin; Jones, David T.W.; Hartmann, Christian; Felsberg, Joerg; Platten, Michael; Feiden, Wolfgang; Keyvani, Kathy; Pfister, Stefan M.; Wiestler, Otmar D.; Meyermann, Richard; Reifenberger, Guido; Pietsch, Thorsten; von Deimling, Andreas; Weller, Michael; Wick, Wolfgang

2014-01-01

Background Molecular biomarkers including isocitrate dehydrogenase 1 or 2 (IDH1/2) mutation, 1p/19q codeletion, and O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation may improve prognostication and guide treatment decisions for patients with World Health Organization (WHO) anaplastic gliomas. At present, each marker is individually tested by distinct assays. Illumina Infinium HumanMethylation450 BeadChip arrays (HM450) enable the determination of large-scale methylation profiles and genome-wide DNA copy number changes. Algorithms have been developed to detect the glioma CpG island methylator phenotype (G-CIMP) associated with IDH1/2 mutation, 1p/19q codeletion, and MGMT promoter methylation using a single assay. Methods Here, we retrospectively investigated the diagnostic and prognostic performance of these algorithms in comparison to individual marker testing and patient outcome in the biomarker cohort (n = 115 patients) of the NOA-04 trial. Results Concordance for IDH and 1p/19q status was very high: In 92% of samples, the HM450 and reference data agreed. In discordant samples, survival analysis by Kaplan-Meier and Cox regression analyses suggested a more accurate assessment of biological phenotype by the HM450 analysis. The HM450-derived MGMT-STP27 model to calculate MGMT promoter methylation probability revealed this aberration in a significantly higher fraction of samples than conventional methylation-specific PCR, with 87 of 91 G-CIMP tumors predicted as MGMT promoter-methylated. Pyrosequencing of discordant samples confirmed the HM450 assessment in 14 of 17 cases. Conclusions G-CIMP and 1p/19q codeletion are reliably detectable by HM450 analysis and are associated with prognosis in the NOA-04 trial. For MGMT, HM450 suggests promoter methylation in the vast majority of G-CIMP tumors, which is supported by pyrosequencing. PMID:25028501

MGMT expression levels predict disease stabilisation, progression-free and overall survival in patients with advanced melanomas treated with DTIC.

PubMed

Busch, Christian; Geisler, Jürgen; Lillehaug, Johan R; Lønning, Per Eystein

2010-07-01

Metastatic melanoma responds poorly to systemic treatment. We report the results of a prospective single institution study evaluating O(6)-methylguanine-DNA methyltransferase (MGMT) status as a potential predictive and/or prognostic marker among patients treated with dacarbazine (DTIC) 800-1000 mg/m(2) monotherapy administered as a 3-weekly schedule for advanced malignant melanomas. The study was approved by the Regional Ethical Committee. Surgical biopsies from metastatic or loco-regional deposits obtained prior to DTIC treatment were snap-frozen immediately upon removal and stored in liquid nitrogen up to processing. Median time from enrolment to end of follow-up was 67 months. MGMT expression levels evaluated by qRT-PCR correlated significantly to DTIC benefit (CR/PR/SD; p=0.005), time to progression (TTP) (p=0.005) and overall survival (OS) (p=0.003). MGMT expression also correlated to Breslow thickness in the primary tumour (p=0.014). While MGMT promoter hypermethylation correlated to MGMT expression, MGMT promoter hypermethylation did not correlate to treatment benefit, TTP or OS, suggesting that other factors may be critical in determining MGMT expression levels in melanomas. In a Cox proportional regression analysis, serum lactate dehydrogenase (LDH, p<0.001), MGMT expression (p=0.022) and p16(INK4a) expression (p=0.037) independently predicted OS, while TTP correlated to DTIC benefit after 6 weeks only (p=0.001). Our data reveal MGMT expression levels to be associated with disease stabilisation and prognosis in patients receiving DTIC monotherapy for advanced melanoma. The role of MGMT expression as a predictor to DTIC sensitivity versus a general prognostic factor in advanced melanomas warrants further evaluation. Copyright 2010 Elsevier Ltd. All rights reserved.

EG-11DYSREGULATION OF MGMT IN GLIOBLASTOMA: FRIEND OR FOE?

PubMed Central

Rapkins, Robert W.; Hitchins, Megan P.; McDonald, Kerrie L.

2014-01-01

Glioblastoma (GBM) is the most common and lethal form of brain cancer (median survival <15 months). The DNA alkylating agent, temozolomide, is used as the standard chemotherapeutic agent, resulting in mispairing of guanine with thymidine that leads to cellular arrest. However, in GBM patients the O6-methylguanine-DNA methyltransferase (MGMT) protein protects DNA from damage induced by temozolomide. Nevertheless, loss of MGMT expression is a frequent event in human malignancies and typically the result of MGMT promoter methylation. MGMT methylation has been strongly associated with the T-allele of the rs16906252 SNP (C/T) in colorectal carcinoma, pleural mesothelioma, and lung cancers. We therefore examined the T-allele and MGMT methylation in temozolmide-treated GBM patients. In 255 temozolomide-treated GBM patients, we found that the T-allele was significantly more frequent in patients with a methylated MGMT promoter. The unadjusted hazard ratio for death in carriers of the T-allele compared to wild-type, irrespective of methylation status, was 0.39 (95%CI:0.21-0.73; p = 0.003), indicating a 61% relative reduction in the risk for death of T-allele carriers. Surprisingly, GBM patients harboring the T-allele in the absence of MGMT methylation showed a survival benefit comparable to those with MGMT methylation (median survival: 15.5 months) and significantly better than the median survival of wild-type, unmethylated patients (median survival: 10.3 months). This suggests that the T-allele may reduce MGMT activity by mechanisms independent of methylation. Genotyping of 451 healthy controls indicated the frequency of carriage of the T-allele was 13% (MAF 0.065). In contrast, carriage of the T-allele in 160 GBM patients was 17%. Significantly, elevated risks were associated with carriage of the T-allele and development of GBM (odds ratio of 2.62 [95%CI:1.7-4.2]). We report that the T-allele (rs16906252) has predictive (response to temozolomide) and prognostic value (MGMT

Residual Deep Convolutional Neural Network Predicts MGMT Methylation Status.

PubMed

Korfiatis, Panagiotis; Kline, Timothy L; Lachance, Daniel H; Parney, Ian F; Buckner, Jan C; Erickson, Bradley J

2017-10-01

Predicting methylation of the O6-methylguanine methyltransferase (MGMT) gene status utilizing MRI imaging is of high importance since it is a predictor of response and prognosis in brain tumors. In this study, we compare three different residual deep neural network (ResNet) architectures to evaluate their ability in predicting MGMT methylation status without the need for a distinct tumor segmentation step. We found that the ResNet50 (50 layers) architecture was the best performing model, achieving an accuracy of 94.90% (+/- 3.92%) for the test set (classification of a slice as no tumor, methylated MGMT, or non-methylated). ResNet34 (34 layers) achieved 80.72% (+/- 13.61%) while ResNet18 (18 layers) accuracy was 76.75% (+/- 20.67%). ResNet50 performance was statistically significantly better than both ResNet18 and ResNet34 architectures (p < 0.001). We report a method that alleviates the need of extensive preprocessing and acts as a proof of concept that deep neural architectures can be used to predict molecular biomarkers from routine medical images.

Can high-dose fotemustine reverse MGMT resistance in glioblastoma multiforme?

PubMed

Gallo, Chiara; Buonerba, Carlo; Di Lorenzo, Giuseppe; Romeo, Valeria; De Placido, Sabino; Marinelli, Alfredo

2010-11-01

Glioblastoma multiforme (GBM), the highest grade malignant glioma, is associated with a grim prognosis-median overall survival is in the range 12-15 months, despite optimum treatment. Surgery to the maximum possible extent, external beam radiotherapy, and systemic temozolomide chemotherapy are current standard treatments for newly diagnosed GBM, with intracerebral delivery of carmustine wafers (Gliadel). Unfortunately, the effectiveness of chemotherapy can be hampered by the DNA repair enzyme O6-methylguanine methyltransferase (MGMT), which confers resistance both to temozolomide and nitrosoureas, for example fotemustine and carmustine. MGMT activity can be measured by PCR and immunohistochemistry, with the former being the current validated technique. High-dose chemotherapy can deplete MGMT levels in GBM cells and has proved feasible in various trials on temozolomide, in both newly diagnosed and recurrent GBM. We here report the unique case of a GBM patient, with high MGMT expression by immunohistochemistry, who underwent an experimental, high-dose fotemustine schedule after surgery and radiotherapy. Although treatment caused two episodes of grade 3-4 thrombocytopenia, a complete response and survival of more than three years were achieved, with a 30% increase in dose intensity compared with the standard fotemustine schedule.

Methylation and expression profiles of MGMT gene in thymic epithelial tumors.

PubMed

Mokhtar, Mohamed; Kondo, Kazuya; Namura, Toshiaki; Ali, Abdellah H K; Fujita, Yui; Takai, Chikako; Takizawa, Hiromitsu; Nakagawa, Yasushi; Toba, Hiroaki; Kajiura, Koichiro; Yoshida, Mitsuteru; Kawakami, Gyokei; Sakiyama, Shoji; Tangoku, Akira

2014-02-01

A key challenge in diagnosis and treatment of thymic epithelial tumors (TET) is in improving our understanding of the genetic and epigenetic changes of these relatively rare tumors. Methylation specific PCR (MSP) and immunohistochemistry were applied to 66 TET to profile the methylation status of DNA repair gene O6-methylguanine DNA methyltransferase (MGMT) and its protein expression in TET to clarify the association between MGMT status and clinicopathological features, response to chemotherapy and overall survival. MGMT methylation was significantly more frequent in thymic carcinoma than in thymoma (17/23, 74% versus 13/44, 29%; P<0.001). Loss of expression of MGMT protein was significantly more frequent in thymic carcinoma than in thymoma (20/23, 87% versus 10/44, 23%; P<0.0001). There is a significant correlation between of MGMT methylation and loss of its protein expression (P<0.0003). MGMT methylation and loss of expression were significantly more frequent in advanced thymic epithelial tumors (III/IV) than in early tumors (I/II). MGMT methylation plays a soul role in development of TET, especially in thymic carcinoma. Therefore, translation of our results from basic molecular research to clinical practice may have important implication for considering MGMT methylation as a marker and a target of future therapies in TET. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A methionine-free diet associated with nitrosourea treatment down-regulates methylguanine-DNA methyl transferase activity in patients with metastatic cancer.

PubMed

Thivat, Emilie; Durando, Xavier; Demidem, Aïcha; Farges, Marie-Chantal; Rapp, Maryse; Cellarier, Eric; Guenin, Samuel; D'Incan, Michel; Vasson, Marie-Paule; Chollet, Philippe

2007-01-01

Methionine (MET) depletion used in association with chemotherapy improves the therapeutic index in animal models. This potentiating effect may be due to tumor cell sensitization to chloroethylnitrosoureas through their MET dependency and the down-regulation of O6- methylguanine-DNA methyltransferase (MGMT). Our purpose was to evaluate the impact of the association of a dietary MET restriction with nitrosourea treatment on MGMT activity in peripheral blood mononuclear cells (PBMCs). Six patients with metastatic cancer (melanoma and glioma) received 4 cycles of a MET-free diet with cystemustine (60 mg/m2). MGMT activity in PBMCs decreased by an average of 13% from 553+/-90 fnol/mg before the diet to 413+/-59 fmol/mg after the diet + chemotherapy period (p=0.029). The decrease of MGMT activity was not affected by the duration of the MET-free diet period but seems to be correlated to the plasma MET depletion induced by the MET-free diet.

MGMT hypomethylation is associated with DNA damage in workers exposed to low-dose benzene.

PubMed

Li, Jie; Zhang, Xinjie; He, Zhini; Sun, Qing; Qin, Fei; Huang, Zhenlie; Zhang, Xiao; Sun, Xin; Liu, Linhua; Chen, Liping; Gao, Chen; Wang, Shan; Wang, Fangping; Li, Daochuan; Zeng, Xiaowen; Deng, Qifei; Wang, Qing; Zhang, Bo; Tang, Huanwen; Chen, Wen; Xiao, Yongmei

2017-07-01

This study aims to assess the effects of low-dose benzene on DNA damage and O 6 -methylguanine-DNA methyltransferase (MGMT) methylation in occupational workers. We recruited 96 nonsmoking male petrochemical industry workers exposed to low-dose benzene and 100 matched control workers. Urinary S-phenylmercapturic acid (SPMA) and S-benzylmercapturic acid (SBMA) were measured for indicating internal exposure of benzene and toluene. The degree of DNA damage was determined by the Comet assay. The levels of MGMT methylation were detected quantitatively by bisulphite-PCR pyrosequencing assay. The benzene-exposed workers had significantly higher levels of urinary SPMA, degree of DNA damage but decreased MGMT methylation than the controls (all p < 0.05). In contrast, the level of urinary SBMA does not differ between benzene-exposed workers and the controls. In all participants, MGMT methylation was negatively associated with the urinary SPMA and the degree of DNA damage, indicating that epigenetic regulation might be involved in response to low-dose benzene exposure-induced genetic damage. MGMT methylation could be a potent biomarker associated with low-dose benzene exposure and benzene-induced DNA damage.

[p16 and MGMT gene methylation in sputum cells of uranium workers].

PubMed

Su, Shi-biao; Yang, Lu-jing; Zhang, Wei; Jin, Ya-li; Nie, Ji-hua; Tong, Jian

2006-02-01

To study the methylation of O-6-methylguanine-DNA methyltransferase (MGMT) and p16 gene in the sputum cells of radon-exposed population. To provide the experimental base for finding the molecular biomarker of the high risk population of the radon-induced lung cancer. 91 radon-exposed workers were divided into 4 groups, high dosage group (> 120 WLM), middle dosage group (between 60 and 120 WLM), low dosage group (between 30 and 60 WLB) and lower dosage group (between 2 and 30 WLM) according to the accumulated exposure dosage of the radon daughters. The abnormal methylation of p16 and MGMT gene in the sputum cells of the population in the four groups was detected with the methylation specific PCR (MSP). There was significantly upward trend for the p16 gene methylation rate (0.00%-20.00%), the MGMT gene methylation rate (0.00%-28.00%) and the total methylation rate (0.00%-40.00%) with the increase of the accumulated exposure dosage of the radon daughters (P < 0.01). The methylation of p16 and MGMT gene is related to the accumulate exposure dosage of the radon daughters.

Digital PCR assessment of MGMT promoter methylation coupled with reduced protein expression optimises prediction of response to alkylating agents in metastatic colorectal cancer patients.

PubMed

Sartore-Bianchi, Andrea; Pietrantonio, Filippo; Amatu, Alessio; Milione, Massimo; Cassingena, Andrea; Ghezzi, Silvia; Caporale, Marta; Berenato, Rosa; Falcomatà, Chiara; Pellegrinelli, Alessio; Bardelli, Alberto; Nichelatti, Michele; Tosi, Federica; De Braud, Filippo; Di Nicolantonio, Federica; Barault, Ludovic; Siena, Salvatore

2017-01-01

O(6)-methylguanine-DNA-methyltransferase (MGMT) is a repair protein, and its deficiency makes tumours more susceptible to the cytotoxic effect of alkylating agents. Five clinical trials with temozolomide or dacarbazine have been performed in metastatic colorectal cancer (mCRC) with selection based on methyl-specific PCR (MSP) testing with modest results. We hypothesised that mitigated results are consequences of unspecific patient selection and that alternative methodologies for MGMT testing such as immunohistochemistry (IHC) and digital polymerase chain reaction (PCR) could enhance patient enrolment. Formalin-fixed paraffin embedded archival tumour tissue samples from four phase II studies of temozolomide or dacarbazine in MGMT MSP-positive mCRCs were analysed by IHC for MGMT protein expression and by methyl-BEAMing (MB) for percentage of promoter methylation. Pooled data were then retrospectively analysed according to objective response rate, progression-free survival (PFS) and overall survival (OS). One hundred and five patients were included in the study. Twelve had achieved partial response (PR) (11.4%), 24 stable disease (SD; 22.9%) and 69 progressive disease (PD; 65.7%). Patients with PR/SD had lower IHC scores and higher MB levels than those with PD. MGMT expression by IHC was negatively and MB levels positively associated with PFS (p < 0.001 and 0.004, respectively), but not with OS. By combining both assays, IHC low/MB high patients displayed an 87% reduction in the hazard of progression (p < 0.001) and a 77% in the hazard for death (p = 0.001). In mCRC selected for MGMT deficiency by MSP, IHC and MB testing improve clinical outcome to alkylating agents. Their combination could enhance patient selection in this setting. Copyright © 2016 Elsevier Ltd. All rights reserved.

Procarbazine and CCNU Chemotherapy for Recurrent Glioblastoma with MGMT Promoter Methylation.

PubMed

Kim, Se-Hyuk; Yoo, Heon; Chang, Jong Hee; Kim, Chae-Yong; Chung, Dong Sup; Kim, Se Hoon; Park, Sung-Hae; Lee, Youn Soo; Yang, Seung Ho

2018-06-11

While procarbazine, CCNU (lomustine), and vincristine (PCV) has been an alternative chemotherapy option for malignant gliomas, it is worth investigating whether the combination of only procarbazine and CCNU is comparable because vincristine adds toxicity with uncertain benefit. The purpose of this study was to evaluate the feasibility of procarbazine and CCNU chemotherapy for recurrent glioblastoma multiforme (GBM) with O 6 -methylguanine-DNA-methyltransferase (MGMT) promoter methylation. Eight patients with recurrent GBM following concurrent chemoradiotherapy and temozolomide (TMZ) adjuvant therapy were enrolled in this trial; they received no other chemotherapeutic agents or target therapy. They received CCNU (75 mg/m 2 ) on day 1 and procarbazine (60 mg/m 2 ) through days 11 and 24 every 4 weeks. The median cycle of CCNU and procarbazine was 3.5 (range: 2-6). One patient achieved stable disease. The median progression-free survival (PFS) with procarbazine and CCNU chemotherapy was eight weeks (range: 5-73), and the PFS rates were 25% and 12.5% at 16 and 30 weeks, respectively. The median overall survival (OS) from the initial diagnosis to death was 40 months, and the median OS from the administration of procarbazine and CCNU chemotherapy to death was 9.7 months (95% confidence interval: 6.7-12.7). Serious adverse events were found at six visits, and two cases were considered to be grade 3 toxicities. The efficacy of procarbazine and CCNU chemotherapy is not satisfactory. This study suggests the need to develop other treatment strategies for recurrent and TMZ-refractory GBM. Trial registry at ClinicalTrials.gov, NCT017337346.

Bayesian inference supports a location and neighbour-dependent model of DNA methylation propagation at the MGMT gene promoter in lung tumours.

PubMed

Bonello, Nicolas; Sampson, James; Burn, John; Wilson, Ian J; McGrown, Gail; Margison, Geoff P; Thorncroft, Mary; Crossbie, Philip; Povey, Andrew C; Santibanez-Koref, Mauro; Walters, Kevin

2013-11-07

We exploit model-based Bayesian inference methodologies to analyse lung tumour-derived methylation data from a CpG island in the O6-methylguanine-DNA methyltransferase (MGMT) promoter. Interest is in modelling the changes in methylation patterns in a CpG island in the first exon of the promoter during lung tumour development. We propose four competils of methylation state propagation based on two mechanisms. The first is the location-dependence mechanism in which the probability of a gain or loss of methylation at a CpG within the promoter depends upon its location in the CpG sequence. The second mechanism is that of neighbour-dependence in which gain or loss of methylation at a CpG depends upon the methylation status of the immediately preceding CpG. Our data comprises the methylation status at 12 CpGs near the 5' end of the CpG island in two lung tumour samples for both alleles of a nearby polymorphism. We use approximate Bayesian computation, a computationally intensive rejection-sampling algorithm to infer model parameters and compare models without the need to evaluate the likelihood function. We compare the four proposed models using two criteria: the approximate Bayes factors and the distribution of the Euclidean distance between the summary statistics of the observed and simulated datasets. Our model-based analysis demonstrates compelling evidence for both location and neighbour dependence in the process of aberrant DNA methylation of this MGMT promoter CpG island in lung tumours. We find equivocal evidence to support the hypothesis that the methylation patterns of the two alleles evolve independently. © 2013 Published by Elsevier Ltd. All rights reserved.

Formation and degradation of nitrogen mustard-induced MGMT-DNA crosslinking in 16HBE cells.

PubMed

Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Zhao, Jiqing; Zou, Zhongmin

2017-08-15

N-methyl-2,2-di(chloroethyl)amine (HN2) is a kind of bifunctional alkyltating agent, which can react with nucleophilic groups in DNA and/or protein to form HN2-bridged crosslinking of target molecules, such as DNA-protein crosslinkings (DPC). O 6 -methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme which solely repairs alkyl adduct on DNA directly. However, MGMT was detected to act as a protein cross-linked with DNA via alkylation in presence of HN2, and unexpectedly turned into a DNA damage enhancer in the form of MGMT-DNA cross-link (mDPC). Present study aimed to explore the possible ways to lessen the incorporation of MGMT into DPC as well as to save it for DNA repair. To find out the influencing factors of mDPC formation and cleavage, human bronchial epithelial cell line 16HBE was exposed to HN2 and the factors related with MGMT expression and degradation were investigated. When c-Myc, a negative transcriptional factor of MGMT was inhibited by 10058-F4, MGMT expression and mDPC formation were increased, and more γ-H2AX was also detected. Sustained treatment with O 6 BG, a specific exogenous substrate and depleter of MGMT, could reduce the level of MGMT and mDPC formation. In contrast, a transient 1h pre-treatment of O 6 GB before HN2 exposure would cause a high MGMT and mDPC level. MGMT was increasingly ubiquitinated after HN2 exposure in a time-dependent manner. At the same time, MGMT was also SUMOylated with a downward time-dependent manner compared to its ubiquitination. Inhibitors of E1, E2 or E3 ligases of ubiqutination all led to the accumulation of mDPC and total-DPC (tDPC) with the difference as that mDPC was sensitive to E1 inhibitor while tDPC more sensitive to E2 and E3 inhibitor. Our results demonstrated the control of mDPC level could be realized through transcription inhibitory effect of c-Myc, O 6 GB application, and the acceleration of mDPC ubiquitination and subsequent degradation. Copyright © 2017. Published by Elsevier

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase.

PubMed

Cornetta, K; Croop, J; Dropcho, E; Abonour, R; Kieran, M W; Kreissman, S; Reeves, L; Erickson, L C; Williams, D A

2006-09-01

Administration of chemotherapy is often limited by myelosuppression. Expression of drug-resistance genes in hematopoietic cells has been proposed as a means to decrease the toxicity of cytotoxic agents. In this pilot study, we utilized a retroviral vector expressing methylguanine DNA methyltransferase (MGMT) to transduce hematopoietic progenitors, which were subsequently used in the setting of alkylator therapy (procarbazine, CCNU, vincristine (PCV)) for poor prognosis brain tumors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells were collected by apheresis and enriched for CD34+ expression. Nine subjects were infused with CD34+-enriched cells treated in a transduction procedure involving a 4-day exposure to cytokines with vector exposure on days 3 and 4. No major adverse event was related to the gene therapy procedure. Importantly, the engraftment kinetics of the treated product was similar to unmanipulated peripheral blood stem cells, suggesting that the ex vivo manipulation did not significantly reduce engrafting progenitor cell function. Gene-transduced cells were detected in all subjects. Although the level and duration was limited, patients receiving cells transduced using fibronectin 'preloaded' with virus supernatant appeared to show improved in vivo marking frequency. These findings demonstrate the feasibility and safety of utilizing MGMT-transduced CD34+ peripheral blood progenitor cells in the setting of chemotherapy.

MGMT Gene Promoter Methylation as a Potent Prognostic Factor in Glioblastoma Treated With Temozolomide-Based Chemoradiotherapy: A Single-Institution Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Young Suk; Kim, Se Hoon; Cho, Jaeho

2012-11-01

Purpose: Recently, cells deficient in O{sup 6}-methylguanine-DNA methyltransferase (MGMT) were found to show increased sensitivity to temozolomide (TMZ). We evaluated whether hypermethylation of MGMT was associated with survival in patients with glioblastoma multiforme (GBM). Methods and Materials: We retrospectively analyzed 93 patients with histologically confirmed GBM who received involved-field radiotherapy with TMZ from 2001 to 2008. The median age was 58 years (range, 24-78 years). Surgical resection was total in 39 patients (42%), subtotal in 30 patients (32%), and partial in 17 patients (18%); only a biopsy was performed in 7 patients (8%). Postoperative radiotherapy began within 3 weeks ofmore » surgery in 87% of the patients. Radiotherapy doses ranged from 50 to 74 Gy (median, 70 Gy). MGMT gene methylation was determined in 78 patients; MGMT was unmethylated in 43 patients (55%) and methylated in 35 patients (45%). The median follow-up period was 22 months (range, 3-88 months) for all patients. Results: The median overall survival (OS) was 22 months, and progression-free survival (PFS) was 11 months. MGMT gene methylation was an independently significant prognostic factor for both OS (p = 0.002) and PFS (p = 0.008) in multivariate analysis. The median OS was 29 months for the methylated group and 20 months for the unmethylated group. In 35 patients with methylated MGMT genes, the 2-year and 5-year OS rates were 54% and 31%, respectively. Six patients with combined prognostic factors of methylated MGMT genes, age {<=}50 years, and total/subtotal resections are all alive 38 to 77 months after operation, whereas the median OS in 8 patients with unmethylated MGMT genes, age >50 years, and less than subtotal resection was 13.2 months. Conclusion: We confirmed that MGMT gene methylation is a potent prognostic factor in patients with GBM. Our results suggest that early postoperative radiotherapy and a high total/subtotal resection rate might further improve

MRI to MGMT: predicting methylation status in glioblastoma patients using convolutional recurrent neural networks

PubMed Central

Han, Lichy; Kamdar, Maulik R.

2017-01-01

Glioblastoma Multiforme (GBM), a malignant brain tumor, is among the most lethal of all cancers. Temozolomide is the primary chemotherapy treatment for patients diagnosed with GBM. The methylation status of the promoter or the enhancer regions of the O6− methylguanine methyltransferase (MGMT) gene may impact the efficacy and sensitivity of temozolomide, and hence may affect overall patient survival. Microscopic genetic changes may manifest as macroscopic morphological changes in the brain tumors that can be detected using magnetic resonance imaging (MRI), which can serve as noninvasive biomarkers for determining methylation of MGMT regulatory regions. In this research, we use a compendium of brain MRI scans of GBM patients collected from The Cancer Imaging Archive (TCIA) combined with methylation data from The Cancer Genome Atlas (TCGA) to predict the methylation state of the MGMT regulatory regions in these patients. Our approach relies on a bi-directional convolutional recurrent neural network architecture (CRNN) that leverages the spatial aspects of these 3-dimensional MRI scans. Our CRNN obtains an accuracy of 67% on the validation data and 62% on the test data, with precision and recall both at 67%, suggesting the existence of MRI features that may complement existing markers for GBM patient stratification and prognosis. We have additionally presented our model via a novel neural network visualization platform, which we have developed to improve interpretability of deep learning MRI-based classification models. PMID:29218894

PAM-OBG: A monoamine oxidase B specific prodrug that inhibits MGMT and generates DNA interstrand crosslinks, potentiating temozolomide and chemoradiation therapy in intracranial glioblastoma

PubMed Central

Sharpe, Martyn A.; Raghavan, Sudhir; Baskin, David S.

2018-01-01

Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O6-methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome. We have designed and tested both in vitro and in vivo, a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O6-benzylguanine (O6BG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to O6BG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER). In mouse intracranial models of primary human glioma, we show that PAM-OBG increases survival of mice treated with either BCNU or CCNU by a factor of six and that in a chemoradiation model utilizing six rounds of TMZ/2Gy radiation, pre-treatment with PAM-OBG more than doubled survival time. PMID:29844863

PAM-OBG: A monoamine oxidase B specific prodrug that inhibits MGMT and generates DNA interstrand crosslinks, potentiating temozolomide and chemoradiation therapy in intracranial glioblastoma.

PubMed

Sharpe, Martyn A; Raghavan, Sudhir; Baskin, David S

2018-05-08

Via extensive analyses of genetic databases, we have characterized the DNA-repair capacity of glioblastoma with respect to patient survival. In addition to elevation of O 6 -methylguanine DNA methyltransferase (MGMT), down-regulation of three DNA repair pathways; canonical mismatch repair (MMR), Non-Homologous End-Joining (NHEJ), and Homologous Recombination (HR) are correlated with poor patient outcome. We have designed and tested both in vitro and in vivo , a monoamine oxidase B (MAOB) specific prodrug, PAM-OBG, that is converted by glioma MAOB into the MGMT inhibitor O 6 -benzylguanine (O 6 BG) and the DNA crosslinking agent acrolein. In cultured glioma cells, we show that PAM-OBG is converted to O 6 BG, inhibiting MGMT and sensitizing cells to DNA alkylating agents such as BCNU, CCNU, and Temozolomide (TMZ). In addition, we demonstrate that the acrolein generated is highly toxic in glioma treated with an inhibitor of Nucleotide Excision Repair (NER). In mouse intracranial models of primary human glioma, we show that PAM-OBG increases survival of mice treated with either BCNU or CCNU by a factor of six and that in a chemoradiation model utilizing six rounds of TMZ/2Gy radiation, pre-treatment with PAM-OBG more than doubled survival time.

MGMT methylation analysis of glioblastoma on the Infinium methylation BeadChip identifies two distinct CpG regions associated with gene silencing and outcome, yielding a prediction model for comparisons across datasets, tumor grades, and CIMP-status.

PubMed

Bady, Pierre; Sciuscio, Davide; Diserens, Annie-Claire; Bloch, Jocelyne; van den Bent, Martin J; Marosi, Christine; Dietrich, Pierre-Yves; Weller, Michael; Mariani, Luigi; Heppner, Frank L; Mcdonald, David R; Lacombe, Denis; Stupp, Roger; Delorenzi, Mauro; Hegi, Monika E

2012-10-01

The methylation status of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is an important predictive biomarker for benefit from alkylating agent therapy in glioblastoma. Recent studies in anaplastic glioma suggest a prognostic value for MGMT methylation. Investigation of pathogenetic and epigenetic features of this intriguingly distinct behavior requires accurate MGMT classification to assess high throughput molecular databases. Promoter methylation-mediated gene silencing is strongly dependent on the location of the methylated CpGs, complicating classification. Using the HumanMethylation450 (HM-450K) BeadChip interrogating 176 CpGs annotated for the MGMT gene, with 14 located in the promoter, two distinct regions in the CpG island of the promoter were identified with high importance for gene silencing and outcome prediction. A logistic regression model (MGMT-STP27) comprising probes cg12434587 [corrected] and cg12981137 provided good classification properties and prognostic value (kappa = 0.85; log-rank p < 0.001) using a training-set of 63 glioblastomas from homogenously treated patients, for whom MGMT methylation was previously shown to be predictive for outcome based on classification by methylation-specific PCR. MGMT-STP27 was successfully validated in an independent cohort of chemo-radiotherapy-treated glioblastoma patients (n = 50; kappa = 0.88; outcome, log-rank p < 0.001). Lower prevalence of MGMT methylation among CpG island methylator phenotype (CIMP) positive tumors was found in glioblastomas from The Cancer Genome Atlas than in low grade and anaplastic glioma cohorts, while in CIMP-negative gliomas MGMT was classified as methylated in approximately 50 % regardless of tumor grade. The proposed MGMT-STP27 prediction model allows mining of datasets derived on the HM-450K or HM-27K BeadChip to explore effects of distinct epigenetic context of MGMT methylation suspected to modulate treatment resistance in different tumor types.

SNP rs16906252C>T is an expression and methylation quantitative trait locus associated with an increased risk of developing MGMT-methylated colorectal cancer

PubMed Central

Kuroiwa-Trzmielina, Joice; Wang, Fan; Rapkins, Robert W.; Ward, Robyn L.; Buchanan, Daniel D.; Win, Aung Ko; Clendenning, Mark; Rosty, Christophe; Southey, Melissa C.; Winship, Ingrid M.; Hopper, John L.; Jenkins, Mark A.; Olivier, Jake; Hawkins, Nicholas J.; Hitchins, Megan P.

2016-01-01

Purpose Methylation of the MGMT promoter is the major cause of O6-methylguanine methyltransferase deficiency in cancer and has been associated with the T variant of the promoter-enhancer SNP rs16906252C>T. We sought evidence for an association between the rs16906252C>T genotype and increased risk of developing a subtype of colorectal cancer (CRC) featuring MGMT methylation, mediated by genotype-dependent epigenetic silencing within normal tissues. Experimental design By applying a molecular pathological epidemiology case-control study design, associations between rs16906252C>T and risk for CRC overall, and CRC stratified by MGMT methylation status, were estimated using multinomial logistic regression in two independent retrospective series of CRC cases and controls. The test sample comprised 1054 CRC cases and 451 controls from Sydney, Australia. The validation sample comprised 612 CRC cases and 245 controls from the Australasian Colon Cancer Family Registry (ACCFR). To determine if rs16906252C>T was linked to a constitutively altered epigenetic state, quantitative allelic expression and methylation analyses were performed in normal tissues. Results An association between rs16906252C>T and increased risk of developing MGMT-methylated CRC in the Sydney sample was observed (OR 3.3; 95%CI=2.0–5.3; P<0.0001), which was replicated in the ACCFR sample (OR 4.0; 95%CI=2.4–6.8; P<0.0001). The T allele demonstrated ~2.5-fold reduced transcription in normal colorectal mucosa from cases and controls, and was selectively methylated in a minority of normal cells, indicating rs16906252C>T represents an expression and methylation quantitative trait locus. Conclusions We provide evidence that rs16906252C>T is associated with elevated risk for MGMT-methylated CRC, likely mediated by constitutive epigenetic repression of the T allele. PMID:27267851

The effects of tumor treating fields and temozolomide in MGMT expressing and non-expressing patient-derived glioblastoma cells.

PubMed

Clark, Paul A; Gaal, Jordan T; Strebe, Joslyn K; Pasch, Cheri A; Deming, Dustin A; Kuo, John S; Robins, H Ian

2017-02-01

A recent Phase 3 study of newly diagnosed glioblastoma (GBM) demonstrated the addition of tumor treating fields (TTFields) to temozolomide (TMZ) after combined radiation/TMZ significantly increased survival and progression free survival. Preliminary data suggested benefit with both methylated and unmethylated O-6-methylguanine-DNA methyl-transferase (MGMT) promoter status. To date, however, there have been no studies to address the potential interactions of TTFields and TMZ. Thus, the effects of TTFields and TMZ were studied in vitro using patient-derived GBM stem-like cells (GSCs) including MGMT expressing (TMZ resistant: 12.1 and 22GSC) and non-MGMT expressing (TMZ sensitive: 33 and 114GSC) lines. Dose-response curves were constructed using cell proliferation and sphere-forming assays. Results demonstrated a ⩾10-fold increase in TMZ resistance of MGMT-expressing (12.1GSCs: IC 50 =160μM; 22GSCs: IC 50 =44μM) compared to MGMT non-expressing (33GSCs: IC 50 =1.5μM; 114GSCs: IC 50 =5.2μM) lines. TTFields inhibited 12.1 GSC proliferation at all tested doses (50-500kHz) with an optimal frequency of 200kHz. At 200kHz, TTFields inhibited proliferation and tumor sphere formation of both MGMT GSC subtypes at comparable levels (12.1GSC: 74±2.9% and 38±3.2%, respectively; 22GSC: 61±11% and 38±2.6%, respectively; 33GSC: 56±9.5% and 60±7.1%, respectively; 114 GSC: 79±3.5% and 41±4.3%, respectively). In combination, TTFields (200kHz) and TMZ showed an additive anti-neoplastic effect with equal efficacy for TTFields in both cell types (i.e., ± MGMT expression) with no effect on TMZ resistance. This is the first demonstration of the effects of TTFields on cancer stem cells. The expansion of such studies may have clinical implications. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Effects of Tumor Treating Fields and Temozolomide in MGMT Expressing and Non-Expressing Patient-Derived Glioblastoma Cells

PubMed Central

Clark, Paul A.; Gaal, Jordan T; Strebe, Joslyn K.; Pasch, Cheri A; Deming, Dustin A; Kuo, John S.; Robins, H. Ian

2016-01-01

A recent Phase 3 study of newly diagnosed glioblastoma (GBM) demonstrated the addition of Tumor Treating Fields (TTFields) to temozolomide (TMZ) after combined radiation/TMZ significantly increased survival and progression free survival. Preliminary data suggested benefit with both methylated and unmethylated O-6-methylguanine-DNA methyl-transferase (MGMT) promoter status. To date, however, there have been no studies to address the potential interactions of TTFields and TMZ. Thus, the effects of TTFields and TMZ were studied in vitro using patient-derived GBM stem-like cells (GSCs) including MGMT expressing (TMZ resistant:12.1 and 22 GSC) and non-MGMT expressing (TMZ sensitive:33 and 114 GSC) lines. Dose-response curves were constructed using cell proliferation and sphere-forming assays. Results demonstrated a ≥10-fold increase in TMZ resistance of MGMT-expressing (12.1 GSCs: IC50=160 μM; 22 GSCs: IC50=44 μM) compared to MGMT non-expressing (33 GSCs: IC50=1.5 μM; 114 GSCs: IC50=5.2 μM) lines. TTFields inhibited 12.1 GSC proliferation at all tested doses (50-500 kHz) with an optimal frequency of 200 kHz. At 200 kHz, TTFields inhibited proliferation and tumor sphere formation of both MGMT GSC subtypes at comparable levels (12.1 GSC: 74±2.9% and 38±3.2%, respectively; 22 GSC: 61±11% and 38±2.6%, respectively; 33 GSC: 56±9.5% and 60±7.1%, respectively; 114 GSC: 79± 3.5% and 41±4.3%, respectively). In combination, TTFields (200 kHz) and TMZ showed an additive anti-neoplastic effect with equal efficacy for TTFields in both cell types (i.e., +/- MGMT expression) with no effect on TMZ resistance. This is the first demonstration of the effects of TTFields on cancer stem cells. The expansion of such studies may have clinical implications. PMID:27865821

Correlation of MLH1 and MGMT methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients.

PubMed

Li, Xia; Wang, Yibaina; Zhang, Zuoming; Yao, Xiaoping; Ge, Jie; Zhao, Yashuang

2013-11-01

CpG island methylation in the promoter regions of the DNA mismatch repair gene mutator L homologue 1 ( MLH1 ) and DNA repair gene O 6 -methylguanine-DNA methyltransferase ( MGMT ) genes has been shown to occur in the leukocytes of peripheral blood and colorectal tissue. However, it is unclear whether the methylation levels in the blood leukocytes and colorectal tissue are correlated. The present study analyzed and compared the levels of MGMT and MLH1 gene methylation in the leukocytes of peripheral blood and colorectal tissues obtained from patients with colorectal cancer (CRC). The methylation levels of MGMT and MLH1 were examined using methylation-sensitive high-resolution melting (MS-HRM) analysis. A total of 44 patients with CRC were selected based on the MLH1 and MGMT gene methylation levels in the leukocytes of the peripheral blood. Corresponding colorectal tumor and normal tissues were obtained from each patient and the DNA methylation levels were determined. The correlation coefficients were evaluated using Spearman's rank test. Agreement was determined by generalized κ-statistics. Spearman's rank correlation coefficients (r) for the methylation levels of the MGMT and MLH1 genes in the leukocytes of the peripheral blood and normal colorectal tissue were 0.475 and 0.362, respectively (P=0.001 and 0.016, respectively). The agreement of the MGMT and MLH1 gene methylation levels in the leukocytes of the peripheral blood and normal colorectal tissue were graded as fair and poor (κ=0.299 and 0.126, respectively). The methylation levels of MGMT and MLH1 were moderately and weakly correlated between the patient-matched leukocytes and the normal colorectal tissue, respectively. Blood-derived DNA methylation measurements may not always represent the levels of normal colorectal tissue methylation.

Down-regulation of p16 and MGMT promotes the anti-proliferative and pro-apoptotic effects of 5-Aza-dC and radiation on cervical cancer cells.

PubMed

Chen, Guan-di; Qian, De-Ying; Li, Zhi-Gang; Fan, Ge-Ying; You, Ke-Li; Wu, Yi-Long

2017-12-01

Cervical cancer is one of the most common malignancies of the female reproductive system. Therefore, it is critical to investigate the molecular mechanisms involved in the development and progression of cervical cancer. In this study, we stimulated cervical cancer cells with 5-aza-2'-deoxycytidine (5-Aza-dC) and found that this treatment inhibited cell proliferation and induced apoptosis; additionally, methylation of p16 and O-6-methylguanine-DNA methyltransferase (MGMT) was reversed, although their expression was suppressed. 5-Aza-dC inhibited E6 and E7 expression and up-regulated p53, p21, and Rb expression. Cells transfected with siRNAs targeting p16 and MGMT as well as cells stimulated with 5-Aza-dC were arrested in S phase, and the expression of p53, p21, and Rb was up-regulated more significantly. However, when cells were stimulated with 5-Aza-dC after transfection with siRNAs targeting p16 and MGMT, proliferation decreased significantly, and the percentage of cells in the sub-G1 peak and in S phase was significantly increased, suggesting a marked increase in apoptosis. But E6 and E7 overexpression could rescue the observed effects in proliferation. Furthermore, X-ray radiation caused cells to arrest in G2/M phase, but cells transfected with p16- and MGMT-targeted siRNAs followed by X-ray radiation exhibited a significant decrease in proliferation and were shifted toward the sub-G1 peak, also indicating enhanced apoptosis. In addition, the effects of 5-Aza-dC and X-ray radiation were most pronounced when MGMT expression was down-regulated. Therefore, down-regulation of p16 and MGMT expression enhances the anti-proliferative effects of 5-Aza-dC and X-ray radiation. This discovery may provide novel ideas for the treatment of cervical cancer. Copyright © 2017 John Wiley & Sons, Ltd.

Profound prevention of experimental brain metastases of breast cancer by temozolomide in an MGMT-dependent manner.

PubMed

Palmieri, Diane; Duchnowska, Renata; Woditschka, Stephan; Hua, Emily; Qian, Yongzhen; Biernat, Wojciech; Sosińska-Mielcarek, Katarzyna; Gril, Brunilde; Stark, Andreas M; Hewitt, Stephen M; Liewehr, David J; Steinberg, Seth M; Jassem, Jacek; Steeg, Patricia S

2014-05-15

Brain metastases of breast cancer cause neurocognitive damage and are incurable. We evaluated a role for temozolomide in the prevention of brain metastases of breast cancer in experimental brain metastasis models. Temozolomide was administered in mice following earlier injection of brain-tropic HER2-positive JIMT-1-BR3 and triple-negative 231-BR-EGFP sublines, the latter with and without expression of O(6)-methylguanine-DNA methyltransferase (MGMT). In addition, the percentage of MGMT-positive tumor cells in 62 patient-matched sets of breast cancer primary tumors and resected brain metastases was determined immunohistochemically. Temozolomide, when dosed at 50, 25, 10, or 5 mg/kg, 5 days per week, beginning 3 days after inoculation, completely prevented the formation of experimental brain metastases from MGMT-negative 231-BR-EGFP cells. At a 1 mg/kg dose, temozolomide prevented 68% of large brain metastases, and was ineffective at a dose of 0.5 mg/kg. When the 50 mg/kg dose was administered beginning on days 18 or 24, temozolomide efficacy was reduced or absent. Temozolomide was ineffective at preventing brain metastases in MGMT-transduced 231-BR-EGFP and MGMT-expressing JIMT-1-BR3 sublines. In 62 patient-matched sets of primary breast tumors and resected brain metastases, 43.5% of the specimens had concordant low MGMT expression, whereas in another 14.5% of sets high MGMT staining in the primary tumor corresponded with low staining in the brain metastasis. Temozolomide profoundly prevented the outgrowth of experimental brain metastases of breast cancer in an MGMT-dependent manner. These data provide compelling rationale for investigating the preventive efficacy of temozolomide in a clinical setting. ©2014 American Association for Cancer Research.

Poor recognition of O6-isopropyl dG by MGMT triggers double strand break-mediated cell death and micronucleus induction in FANC-deficient cells

PubMed Central

Hashimoto, Kiyohiro; Sharma, Vyom; Sasanuma, Hiroyuki; Tian, Xu; Takata, Minoru; Takeda, Shunichi; Swenberg, James A.; Nakamura, Jun

2016-01-01

Isopropyl methanesulfonate (IPMS) is the most potent genotoxic compound among methanesulfonic acid esters. The genotoxic potential of alkyl sulfonate esters is believed to be due to their alkylating ability of the O6 position of guanine. Understanding the primary repair pathway activated in response to IPMS-induced DNA damage is important to profile the genotoxic potential of IPMS. In the present study, both chicken DT40 and human TK6 cell-based DNA damage response (DDR) assays revealed that dysfunction of the FANC pathway resulted in higher sensitivity to IPMS compared to EMS or MMS. O6-alkyl dG is primarily repaired by methyl guanine methyltransferase (MGMT), while isopropyl dG is less likely to be a substrate for MGMT. Comparison of the cytotoxic potential of IPMS and its isomer n-propyl methanesulfonate (nPMS) revealed that the isopropyl moiety avoids recognition by MGMT and leads to higher cytotoxicity. Next, the micronucleus (MN) assay showed that FANC deficiency increases the sensitivity of DT40 cells to MN induction by IPMS. Pretreatment with O6-benzyl guanine (OBG), an inhibitor of MGMT, increased the MN frequency in DT40 cells treated with nPMS, but not IPMS. Lastly, IPMS induced more double strand breaks in FANC-deficient cells compared to wild-type cells in a time-dependent manner. All together, these results suggest that IPMS-derived O6-isopropyl dG escapes recognition by MGMT, and the unrepaired DNA damage leads to double strand breaks, resulting in MN induction. FANC, therefore, plays a pivotal role in preventing MN induction and cell death caused by IPMS. PMID:27486975

Personalized care in neuro-oncology coming of age: why we need MGMT and 1p/19q testing for malignant glioma patients in clinical practice.

PubMed

Weller, Michael; Stupp, Roger; Hegi, Monika E; van den Bent, Martin; Tonn, Joerg C; Sanson, Marc; Wick, Wolfgang; Reifenberger, Guido

2012-09-01

Histological subtyping and grading by malignancy are the cornerstones of the World Health Organization (WHO) classification of tumors of the central nervous system. They shall provide clinicians with guidance as to the course of disease to be expected and the choices of treatment to be made. Nonetheless, patients with histologically identical tumors may have very different outcomes, notably in patients with astrocytic and oligodendroglial gliomas of WHO grades II and III. In gliomas of adulthood, 3 molecular markers have undergone extensive studies in recent years: 1p/19q chromosomal codeletion, O(6)-methylguanine methyltransferase (MGMT) promoter methylation, and mutations of isocitrate dehydrogenase (IDH) 1 and 2. However, the assessment of these molecular markers has so far not been implemented in clinical routine because of the lack of therapeutic implications. In fact, these markers were considered to be prognostic irrespective of whether patients were receiving radiotherapy (RT), chemotherapy, or both (1p/19q, IDH1/2), or of limited value because testing is too complex and no chemotherapy alternative to temozolomide was available (MGMT). In 2012, this situation has changed: long-term follow-up of the Radiation Therapy Oncology Group 9402 and European Organisation for Research and Treatment of Cancer 26951 trials demonstrated an overall survival benefit from the addition to RT of chemotherapy with procarbazine/CCNU/vincristine confined to patients with anaplastic oligodendroglial tumors with (vs without) 1p/19q codeletion. Furthermore, in elderly glioblastoma patients, the NOA-08 and the Nordic trial of RT alone versus temozolomide alone demonstrated a profound impact of MGMT promoter methylation on outcome by therapy and thus established MGMT as a predictive biomarker in this patient population. These recent results call for the routine implementation of 1p/19q and MGMT testing at least in subpopulations of malignant glioma patients and represent an

Personalized care in neuro-oncology coming of age: why we need MGMT and 1p/19q testing for malignant glioma patients in clinical practice

PubMed Central

Weller, Michael; Stupp, Roger; Hegi, Monika E.; van den Bent, Martin; Tonn, Joerg C.; Sanson, Marc; Wick, Wolfgang; Reifenberger, Guido

2012-01-01

Histological subtyping and grading by malignancy are the cornerstones of the World Health Organization (WHO) classification of tumors of the central nervous system. They shall provide clinicians with guidance as to the course of disease to be expected and the choices of treatment to be made. Nonetheless, patients with histologically identical tumors may have very different outcomes, notably in patients with astrocytic and oligodendroglial gliomas of WHO grades II and III. In gliomas of adulthood, 3 molecular markers have undergone extensive studies in recent years: 1p/19q chromosomal codeletion, O6-methylguanine methyltransferase (MGMT) promoter methylation, and mutations of isocitrate dehydrogenase (IDH) 1 and 2. However, the assessment of these molecular markers has so far not been implemented in clinical routine because of the lack of therapeutic implications. In fact, these markers were considered to be prognostic irrespective of whether patients were receiving radiotherapy (RT), chemotherapy, or both (1p/19q, IDH1/2), or of limited value because testing is too complex and no chemotherapy alternative to temozolomide was available (MGMT). In 2012, this situation has changed: long-term follow-up of the Radiation Therapy Oncology Group 9402 and European Organisation for Research and Treatment of Cancer 26951 trials demonstrated an overall survival benefit from the addition to RT of chemotherapy with procarbazine/CCNU/vincristine confined to patients with anaplastic oligodendroglial tumors with (vs without) 1p/19q codeletion. Furthermore, in elderly glioblastoma patients, the NOA-08 and the Nordic trial of RT alone versus temozolomide alone demonstrated a profound impact of MGMT promoter methylation on outcome by therapy and thus established MGMT as a predictive biomarker in this patient population. These recent results call for the routine implementation of 1p/19q and MGMT testing at least in subpopulations of malignant glioma patients and represent an encouraging

Impact of Therapy Sequence with Alkylating Agents and MGMT Status in Patients with Advanced Neuroendocrine Tumors.

PubMed

Krug, Sebastian; Boch, Michael; Rexin, Peter; Gress, Thomas M; Michl, Patrick; Rinke, Anja

2017-05-01

Alkylating chemotherapeutics with either a streptozotocin-(STZ) or temozolomide-(TEM) backbone are routinely used in patients with progressive and unresectable pancreatic neuroendocrine tumors (PNET). In addition, dacarbazine (DTIC) was described as an alternative alkylating therapy option for PNETs. The optimal treatment sequence with alkylating compounds and a potential use of O6-methylguanine-DNA methyltransferase (MGMT) level as predictive biomarker have not yet been sufficiently elucidated. The aim of our study was the evaluation of therapy sequence with either STZ-based treatment followed by DTIC (group A) or the inverse schedule with upfront DTIC (group B) and to correlate MGMT status with clinicopathological characteristics and response to therapy. We retrospectively analyzed 28 patients with neuroendocrine tumors (NET) who were treated with STZ-based therapy and DTIC. Additionally, in a second group MGMT immunohistochemistry was performed from primary and metastatic tumor sites. For statistical evaluation Kaplan-Meier analysis, Cox regression methods and Fisher's exact test were used. There was no difference of objective response and disease control between either STZ-based therapy followed by DTIC treatment (group A) after progression or the reverse sequence (group B). Median time to progression (TTP) was estimated to be 21 months in both arms. First-line STZ-based chemotherapy was not superior to first-line DTIC treatment (16 vs. 13 months; p=0.8). MGMT status did not correlate with clinicopathological characteristics or response to therapy with these alkylating agents. Upfront chemotherapy with either STZ-based treatment or DTIC monotherapy showed similar efficacy and median TTP rates. In this study, MGMT protein expression assessed by immunohistochemistry did not play an important role as a predictive marker for alkylating agents. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

MGMT Inhibition Restores ERα Functional Sensitivity to Antiestrogen Therapy

PubMed Central

Bobustuc, George C; Smith, Joshua S; Maddipatla, Sreeram; Jeudy, Sheila; Limaye, Arati; Isley, Beth; Caparas, Maria-Lourdes M; Constantino, Susan M; Shah, Nikita; Baker, Cheryl H; Srivenugopal, Kalkunte S; Baidas, Said; Konduri, Santhi D

2012-01-01

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O6 methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O6-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21cip1/waf1. We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21cip1/waf1 and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance. PMID:22549111

Exploring the Roles of Nucleobase Desolvation and Shape Complementarity during the Misreplication of O6-Methylguanine

PubMed Central

Chavarria, Delia; Ramos-Serrano, Andrea; Hirao, Ichiro; Berdis, Anthony J.

2011-01-01

O6-methylguanine is a miscoding DNA lesion arising from the alkylation of guanine. This report uses the bacteriophage T4 DNA polymerase as a model to probe the roles hydrogen-bonding interactions, shape/size, and nucleobase desolvation during the replication of this miscoding lesion. This was accomplished by using transient kinetic techniques to monitor the kinetic parameters for incorporating and extending natural and non-natural nucleotides. In general, the efficiency of nucleotide incorporation does not depend on the hydrogen-bonding potential of the incoming nucleotide. Instead, nucleobase hydrophobicity and shape complementarity appear to be the preeminent factors controlling nucleotide incorporation. In addition, shape complementarity plays a large role in controlling the extension of various mispairs containing O6-methylguanine. This is evident as the rate constants for extension correlate with proper interglycosyl distances and symmetry between the base angles of the formed mispair. Base pairs not conforming to an acceptable geometry within the polymerase’s active site are refractory to elongation and are processed via exonuclease proofreading. The collective data set encompassing nucleotide incorporation, extension, and excision is used to generate a model accounting for the mutagenic potential of O6-methylguanine observed in vivo. In addition, kinetic studies monitoring the incorporation and extension of non-natural nucleotides identified an analog that displays high selectivity for incorporation opposite O6-methylguanine compared to unmodified purines. The unusual selectivity of this analog for replicating damaged DNA provides a novel biochemical tool to study translesion DNA synthesis. PMID:21819995

pH-Sensitive O6-Benzylguanosine Polymer Modified Magnetic Nanoparticles for Treatment of Glioblastomas.

PubMed

Stephen, Zachary R; Gebhart, Rachel N; Jeon, Mike; Blair, Allison A; Ellenbogen, Richard G; Silber, John R; Zhang, Miqin

2017-01-18

Nanoparticle-mediated delivery of chemotherapeutics has demonstrated potential in improving anticancer efficacy by increasing serum half-life and providing tissue specificity and controlled drug release to improve biodistribution of hydrophobic chemotherapeutics. However, suboptimal drug loading, particularly for solid core nanoparticles (NPs), remains a challenge that limits their clinical application. In this study we formulated a NP coated with a pH-sensitive polymer of O 6 -methylguanine-DNA methyltransferase (MGMT) inhibitor analog, dialdehyde modified O 6 -benzylguanosine (DABGS) to achieve high drug loading, and polyethylene glycol (PEG) to ameliorate water solubility and maintain NP stability. The base nanovector consists of an iron oxide core (9 nm) coated with hydrazide functionalized PEG (IOPH). DABGS and PEG-dihydrazide were polymerized on the iron oxide nanoparticle surface (IOPH-pBGS) through acid-labile hydrazone bonds utilizing a rapid, freeze-thaw catalysis approach. DABGS polymerization was confirmed by FTIR and quantitated by UV-vis spectroscopy. IOPH-pBGS demonstrated excellent drug loading of 33.4 ± 5.1% by weight while maintaining small size (36.5 ± 1.8 nm). Drug release was monitored at biologically relevant pHs and demonstrated pH dependent release with maximum release at pH 5.5 (intracellular conditions), and minimal release at physiological pH (7.4). IOPH-pBGS significantly suppressed activity of MGMT and potentiated Temozolomide (TMZ) toxicity in vitro, demonstrating potential as a new treatment option for glioblastomas (GBMs).

Redox-Responsive Magnetic Nanoparticle for Targeted Convection-Enhanced Delivery of O6-Benzylguanine to Brain Tumors

PubMed Central

2015-01-01

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O6-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes. PMID:25247850

Redox-responsive magnetic nanoparticle for targeted convection-enhanced delivery of O6-benzylguanine to brain tumors.

PubMed

Stephen, Zachary R; Kievit, Forrest M; Veiseh, Omid; Chiarelli, Peter A; Fang, Chen; Wang, Kui; Hatzinger, Shelby J; Ellenbogen, Richard G; Silber, John R; Zhang, Miqin

2014-10-28

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O(6)-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes.

Amelioration of murine beta-thalassemia through drug selection of hematopoietic stem cells transduced with a lentiviral vector encoding both gamma-globin and the MGMT drug-resistance gene.

PubMed

Zhao, Huifen; Pestina, Tamara I; Nasimuzzaman, Md; Mehta, Perdeep; Hargrove, Phillip W; Persons, Derek A

2009-06-04

Correction of murine models of beta-thalassemia has been achieved through high-level globin lentiviral vector gene transfer into mouse hematopoietic stem cells (HSCs). However, transduction of human HSCs is less robust and may be inadequate to achieve therapeutic levels of genetically modified erythroid cells. We therefore developed a double gene lentiviral vector encoding both human gamma-globin under the transcriptional control of erythroid regulatory elements and methylguanine methyltransferase (MGMT), driven by a constitutive cellular promoter. MGMT expression provides cellular resistance to alkylator drugs, which can be administered to kill residual untransduced, diseased HSCs, whereas transduced cells are protected. Mice transplanted with beta-thalassemic HSCs transduced with a gamma-globin/MGMT vector initially had subtherapeutic levels of red cells expressing gamma-globin. To enrich gamma-globin-expressing cells, transplanted mice were treated with the alkylator agent 1,3-bis-chloroethyl-1-nitrosourea. This resulted in significant increases in the number of gamma-globin-expressing red cells and the amount of fetal hemoglobin, leading to resolution of anemia. Selection of transduced HSCs was also obtained when cells were drug-treated before transplantation. Mice that received these cells demonstrated reconstitution with therapeutic levels of gamma-globin-expressing cells. These data suggest that MGMT-based drug selection holds promise as a modality to improve gene therapy for beta-thalassemia.

Does the cycad genotoxin MAM implicated in Guam ALS-PDC induce disease-relevant changes in mouse brain that includes olfaction?

PubMed

Kisby, Glen; Palmer, Valerie; Lasarev, Mike; Fry, Rebecca; Iordanov, Mihail; Magun, Eli; Samson, Leona; Spencer, Peter

2011-11-01

Western Pacific amyotrophic lateral sclerosis (ALS) and parkinsonism-dementia complex (PDC), a prototypical neurodegenerative disease (tauopathy) affecting distinct genetic groups with common exposure to neurotoxic chemicals in cycad seed, has many features of Parkinson's and Alzheimer's diseases (AD), including early olfactory dysfunction. Guam ALS-PDC incidence correlates with cycad flour content of cycasin and its aglycone methylazoxymethanol (MAM), which produces persistent DNA damage (O(6)-methylguanine) in the brains of mice lacking O(6)-methylguanine methyltransferase (Mgmt(-/-)). We described in Mgmt(-/-)mice up to 7 days post-MAM treatment that brain DNA damage was linked to brain gene expression changes found in human neurological disease, cancer, and skin and hair development. This addendum reports 6 months post-MAM treatment- related brain transcriptional changes as well as elevated mitogen activated protein kinases and increased caspase-3 activity, both of which are involved in tau aggregation and neurofibrillary tangle formation typical of ALS-PDC and AD, plus transcriptional changes in olfactory receptors. Does cycasin act as a "slow (geno)toxin" in ALS-PDC?

Profound prevention of experimental brain metastases of breast cancer by temozolomide in an MGMT-dependent manner

PubMed Central

Palmieri, Diane; Duchnowska, Renata; Woditschka, Stephan; Hua, Emily; Qian, Yongzhen; Biernat, Wojciech; Sosińska-Mielcarek, Katarzyna; Gril, Brunilde; Stark, Andreas; Hewitt, Stephen; Liewehr, David J; Steinberg, Seth M; Jassem, Jacek; Steeg, Patricia S

2014-01-01

Purpose Brain metastases of breast cancer cause neurocognitive damage and are incurable. We evaluated a role for temozolomide in the prevention of brain metastases of breast cancer in experimental brain metastasis models. Experimental Design Temozolomide was administered in mice following earlier injection of brain-tropic human epidermal growth factor receptor 2 (HER2)-positive Jimt1-BR3 and triple negative 231-BR-EGFP sublines, the latter with and without expression of 06-methylguanine-DNA methyltransferase (MGMT). Additionally, the percentage of MGMT-positive tumor cells in 62 patient-matched sets of breast cancer primary tumors and resected brain metastases was determined immunohistochemically. Results Temozolomide, when dosed at 50, 25, 10 or 5 mg/kg, 5 days/week, beginning 3 days after inoculation, completely prevented the formation of experimental brain metastases from MGMT-negative 231-BR-EGFP cells. At a 1 mg/kg dose, temozolomide prevented 68% of large brain metastases, and was ineffective at a dose of 0.5 mg/kg. When the 50 mg/kg dose was administered beginning on days 18 or 24, temozolomide efficacy was reduced or absent. Temozolomide was ineffective at preventing brain metastases in MGMT-transduced 231-BR-EGFP and MGMT-expressing Jimt-1-BR3 sublines. In 62 patient-matched sets of primary breast tumors and resected brain metastases, 43.5% of the specimens had concordant low MGMT expression, while in another 14.5% of sets high MGMT staining in the primary tumor corresponded with low staining in the brain metastasis. Conclusions Temozolomide profoundly prevented the outgrowth of experimental brain metastases of breast cancer in an MGMT-dependent manner. These data provide compelling rationale for investigating the preventive efficacy of temozolomide in a clinical setting. PMID:24634373

Chemical carcinogenesis in the nervous system. Preferential accumulation of O6-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-N-nitrosourea.

PubMed Central

Margison, G P; Kleihues, P

1975-01-01

The alkylation of purine bases in DNA of several rat tissues was determined during weekly injections (10 mg/kg) of N-[3H]methyl-N-nitrosourea, a dose schedule known to selectively induce tumours of the nervous system. Each group of animals was killed 1 week after the final injection, and the DNA hydrolysates were analysed by chromatography on Sephadex G-10. After five weekly applications, O6-methylguanine had accumulated in brain DNA to an extent which greatly exceeded that in kidney, spleen and intestine. In the liver, the final O6-methylguanine concentration was less than 1% of that in brain. Between the first and the fifth injection, the O6-methylguanine/7-methylguanine ratio in cerebral DNA increased from 0.28 to 0.68. In addition, 3-methylguanine was found to accumulate in brain DNA whereas in the other organs no significant quantities of this base were detectable. The results are compatible with the hypothesis that O6-alkylation of guanine in DNA plays a major role in the induction of tumours by N-methyl-N-nitrosourea and related carcinogens. The kinetics of the increase of O6-methylguanine in cerebral DNA suggest that there is no major cell fraction in the brain which is capable of excising chemically methylated bases from DNA. This repair deficiency could be a determining factor in the selective induction of nervous-system tumours by N-methyl-N-nitrosourea and other neuro-oncogenic compounds. PMID:1200992

Strategy for Imidazotetrazine Prodrugs with Anticancer Activity Independent of MGMT and MMR

PubMed Central

2012-01-01

The imidazotetrazine ring is an acid-stable precursor and prodrug of highly reactive alkyl diazonium ions. We have shown that this reactivity can be managed productively in an aqueous system for the generation of aziridinium ions with 96% efficiency. The new compounds are potent DNA alkylators and have antitumor activity independent of the O6-methylguanine-DNA methyltransferase and DNA mismatch repair constraints that limit the use of Temozolomide. PMID:24900418

Assessing the predictability of IDH mutation and MGMT methylation status in glioma patients using relaxation-compensated multi-pool CEST MRI at 7.0 Tesla.

PubMed

Paech, Daniel; Windschuh, Johannes; Oberhollenzer, Johanna; Dreher, Constantin; Sahm, Felix; Meissner, Jan-Eric; Goerke, Steffen; Schuenke, Patrick; Zaiss, Moritz; Regnery, Sebastian; Bickelhaupt, Sebastian; Bäumer, Philipp; Bendszus, Martin; Wick, Wolfgang; Unterberg, Andreas; Bachert, Peter; Ladd, Mark Edward; Schlemmer, Heinz-Peter; Radbruch, Alexander

2018-05-04

Early identification of prognostic superior characteristics in glioma patients such as Isocitrate dehydrogenase(IDH)-mutation and O6-methylguanine-DNA-methyltransferase (MGMT) promotor methylation status is of great clinical importance. The study purpose was to investigate the non-invasive predictability of IDH-mutation status, MGMT promotor methylation, and differentiation of lower versus higher grade glioma (LGG vs. HGG) in newly-diagnosed patients employing relaxation-compensated multi-pool Chemical Exchange Saturation Transfer (CEST) magnetic resonance imaging (MRI) at 7.0 Tesla (7T). Thirty-one newly-diagnosed glioma patients were included in this prospective study. CEST MRI was performed at a 7T whole-body scanner. Nuclear Overhauser Effect (NOE) and isolated amide proton transfer (APT, downfield NOE-suppressed APT=dns-APT) CEST signals (mean value and 90th signal percentile) were quantitatively investigated in the whole tumor area with regard to predictability of IDH-mutation, MGMT promotor methylation status, and differentiation of LGG vs. HGG. Statistics were performed using receiver operating characteristic (ROC) and area under the curve (AUC) analysis. Results were compared to advanced MRI methods (apparent diffusion coefficient (ADC) and relative cerebral blood volume (rCBV) ROC/AUC analysis) obtained at 3T. dns-APT CEST contrasts yielded highest AUCs in IDH-mutation status prediction (dns-APTmean=91.84%, p<0.01; dns-APT90=97.96%, p<0.001). Furthermore, dns-APT metrics enabled significant differentiation of LGG vs. HGG (AUC: dns-APTmean=0.78, p<0.05; dns-APT90=0.83, p<0.05). There was no significant difference regarding MGMT promotor methylation status at any contrast (p>0.05). Relaxation-compensated multi-pool CEST MRI, particularly dns-APT imaging, enabled prediction of IDH-mutation status and differentiation of LGG vs. HGG and should therefore be considered as non-invasive MR biomarker in the diagnostic workup.

Connexin 43 inhibition sensitizes chemoresistant glioblastoma cells to temozolomide

PubMed Central

Murphy, Susan F; Varghese, Robin T; Lamouille, Samy; Guo, Sujuan; Pridham, Kevin J; Kanabur, Pratik; Osimani, Alyssa M; Sharma, Shaan; Jourdan, Jane; Rodgers, Cara M; Simonds, Gary R; Gourdie, Robert G; Sheng, Zhi

2015-01-01

Resistance of glioblastoma (GBM) to the front-line chemotherapeutic agent temozolomide (TMZ) continues to challenge GBM treatment efforts. The repair of TMZ-induced DNA damage by O-6-methylguanine-DNA methyltransferase (MGMT) confers one mechanism of TMZ resistance. Paradoxically, MGMT-deficient GBM patients survive longer despite still developing resistance to TMZ. Recent studies indicate that the gap junction protein connexin 43 (Cx43) renders GBM cells resistant to TMZ through its carboxyl terminus (CT). In this study, we report insights into how Cx43 promotes TMZ resistance. Cx43 levels were inversely correlated with TMZ sensitivity of GBM cells, including GBM stem cells. Moreover, Cx43 levels inversely correlated with patient survival, including as observed in MGMT-deficient GBM patients. Addition of the C-terminal peptide mimetic αCT1, a selective inhibitor of Cx43 channels, sensitized human MGMT-deficient and TMZ-resistant GBM cells to TMZ treatment. Moreover, combining αCT1 with TMZ blocked AKT/mTOR signaling, induced autophagy and apoptosis in TMZ-resistant GBM cells. Our findings suggest that Cx43 may offer a biomarker to predict the survival of patients with MGMT-independent TMZ resistance, and that combining a Cx43 inhibitor with TMZ could enhance therapeutic responses in GBM and perhaps other TMZ-resistant cancers. PMID:26542214

Oxidative cytotoxic agent withaferin A resensitizes temozolomide-resistant glioblastomas via MGMT depletion and induces apoptosis through Akt/mTOR pathway inhibitory modulation

PubMed Central

Grogan, Patrick T.; Sarkaria, Jann N.; Timmermann, Barbara N.; Cohen, Mark S.

2014-01-01

Temozolomide (TMZ) has remained the chemotherapy of choice in patients with glioblastoma multiforme (GBM) primarily due to the lack of more effective drugs. Tumors, however, quickly develop resistance to this line of treatment creating a critical need for alternative approaches and strategies to resensitize the cells. Withaferin A (WA), a steroidal lactone derived from several genera of the Solanaceae plant family has previously demonstrated potent anti-cancer activity in multiple tumor models. Here, we examine the effects of WA against TMZ-resistant GBM cells as a monotherapy and in combination with TMZ. WA prevented GBM cell proliferation by dose-dependent G2/M cell cycle arrest and cell death through both intrinsic and extrinsic apoptotic pathways. This effect correlated with depletion of principle proteins of the Akt/mTOR and MAPK survival and proliferation pathways with diminished phosphorylation of Akt, mTOR, and p70 S6K but compensatory activation of ERK1/2. Depletion of tyrosine kinase cell surface receptors c-Met, EGFR, and Her2 was also observed. WA demonstrated induction of N-acetyl-L-cysteine-repressible oxidative stress as measured directly and through a subsequent heat shock response with HSP32 and HSP70 upregulation and decreased HSF1. Finally, pretreatment of TMZ-resistant GBM cells with WA was associated with O6-methylguanine-DNA methyltransferase (MGMT) depletion which potentiated TMZ-mediated MGMT degradation. Combination treatment with both WA and TMZ resulted in resensitization of MGMT-mediated TMZ-resistance but not resistance through mismatch repair mutations. These studies suggest great clinical potential for the utilization of WA in TMZ-resistant GBM as both a monotherapy and a resensitizer in combination with the standard chemotherapeutic agent TMZ. PMID:24718901

The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors

PubMed Central

Jaber, Mohammed; Wölfer, Johannes; Ewelt, Christian; Holling, Markus; Hasselblatt, Martin; Niederstadt, Thomas; Zoubi, Tarek; Weckesser, Matthias

2015-01-01

BACKGROUND: Approximately 20% of grade II and most grade III gliomas fluoresce after 5-aminolevulinic acid (5-ALA) application. Conversely, approximately 30% of nonenhancing gliomas are actually high grade. OBJECTIVE: The aim of this study was to identify preoperative factors (ie, age, enhancement, 18F-fluoroethyl tyrosine positron emission tomography [18F-FET PET] uptake ratios) for predicting fluorescence in gliomas without typical glioblastomas imaging features and to determine whether fluorescence will allow prediction of tumor grade or molecular characteristics. METHODS: Patients harboring gliomas without typical glioblastoma imaging features were given 5-ALA. Fluorescence was recorded intraoperatively, and biopsy specimens collected from fluorescing tissue. World Health Organization (WHO) grade, Ki-67/MIB-1 index, IDH1 (R132H) mutation status, O6-methylguanine DNA methyltransferase (MGMT) promoter methylation status, and 1p/19q co-deletion status were assessed. Predictive factors for fluorescence were derived from preoperative magnetic resonance imaging and 18F-FET PET. Classification and regression tree analysis and receiver-operating-characteristic curves were generated for defining predictors. RESULTS: Of 166 tumors, 82 were diagnosed as WHO grade II, 76 as grade III, and 8 as glioblastomas grade IV. Contrast enhancement, tumor volume, and 18F-FET PET uptake ratio >1.85 predicted fluorescence. Fluorescence correlated with WHO grade (P < .001) and Ki-67/MIB-1 index (P < .001), but not with MGMT promoter methylation status, IDH1 mutation status, or 1p19q co-deletion status. The Ki-67/MIB-1 index in fluorescing grade III gliomas was higher than in nonfluorescing tumors, whereas in fluorescing and nonfluorescing grade II tumors, no differences were noted. CONCLUSION: Age, tumor volume, and 18F-FET PET uptake are factors predicting 5-ALA-induced fluorescence in gliomas without typical glioblastoma imaging features. Fluorescence was associated with an increased

Prognosis value of MGMT promoter methylation for patients with lung cancer: a meta-analysis

PubMed Central

Chen, Chao; Hua, Haiqing; Han, Chenglong; Cheng, Yuan; Cheng, Yin; Wang, Zhen; Bao, Jutao

2015-01-01

The role of MGMT promoter methylation in lung cancer (LC) remains controversial. To clarify the association of MGMT promoter methylation with survival in LC, we performed a meta-analysis of the literature with meta-analysis. Trials were selected for further analysis if they provided an independent assessment of MGMT promoter methylation in LC and reported the survival data in the context of MGMT promoter methylation status. Subgroup analyses were conducted according to the study characteristic. A total of 9 trials, which comprised 859 patients, were included in the meta-analysis. The combined hazard ratio (HR) of 1.27 [95% CI 0.88-1.82; test for heterogeneity P = 0.027] suggests that MGMT promoter methylation has none impact on patient survival. In Stage I-III or younger populations, a significant association was found for MGMT promoter methylation in the prognosis of LC. In addition, the heterogeneity disappeared when the analysis was restricted to Stage I-III LC. Our analysis indicates that MGMT promoter methylation in stage I-III or younger patients was significantly correlated with wore survival. Further study is needed to determine these specific subgroups of LC patients. PMID:26617891

Prognosis value of MGMT promoter methylation for patients with lung cancer: a meta-analysis.

PubMed

Chen, Chao; Hua, Haiqing; Han, Chenglong; Cheng, Yuan; Cheng, Yin; Wang, Zhen; Bao, Jutao

2015-01-01

The role of MGMT promoter methylation in lung cancer (LC) remains controversial. To clarify the association of MGMT promoter methylation with survival in LC, we performed a meta-analysis of the literature with meta-analysis. Trials were selected for further analysis if they provided an independent assessment of MGMT promoter methylation in LC and reported the survival data in the context of MGMT promoter methylation status. Subgroup analyses were conducted according to the study characteristic. A total of 9 trials, which comprised 859 patients, were included in the meta-analysis. The combined hazard ratio (HR) of 1.27 [95% CI 0.88-1.82; test for heterogeneity P = 0.027] suggests that MGMT promoter methylation has none impact on patient survival. In Stage I-III or younger populations, a significant association was found for MGMT promoter methylation in the prognosis of LC. In addition, the heterogeneity disappeared when the analysis was restricted to Stage I-III LC. Our analysis indicates that MGMT promoter methylation in stage I-III or younger patients was significantly correlated with wore survival. Further study is needed to determine these specific subgroups of LC patients.

Bifunctional alkylating agent-mediated MGMT-DNA cross-linking and its proteolytic cleavage in 16HBE cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Jin; Ye, Feng; Dan, Guorong

Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O{sup 6}-methylguanine–DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPCmore » was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1 h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24 h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair. - Highlights: • Nitrogen mustard-induced MGMT-DNA cross-linking was detected in a living cell. • Concentration- and time-dependent manners of MGMT-DNA cross-linking were revealed. • Proteolysis played an important role in protein (MGMT)-DNA cross-linking repair. • DVC1 acts as a proteolytic enzyme in cross

The Enzymatic Release of O6-methylguanine and 3-methyladenine from DNA Reacted with the Carcinogen N-methyl-N-nitrosourea

PubMed Central

Kirtikar, D. M.; Goldthwait, D. A.

1974-01-01

Endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) of Escherichia coli has been shown to break phosphodiester bonds in alkylated DNA and depurinated DNA. The hypothesis that depurination is a step in the mechanism of the reaction with alkylated DNA is supported by in vitro experiments with DNA reacted with N-methyl-N-nitrosourea. Endonuclease II releases O6-methylguanine and 3-methyladenine, but not 7-methylguanine, from DNA that has been methylated by the carcinogen N-methyl-N-nitrosourea. PMID:4600266

Expression of DNA mismatch repair proteins MLH1, MSH2, and MSH6 in recurrent glioblastoma.

PubMed

Stark, Andreas M; Doukas, Alexander; Hugo, Heinz-Herrmann; Hedderich, Jürgen; Hattermann, Kirsten; Maximilian Mehdorn, H; Held-Feindt, Janka

2015-02-01

Methylated O6-methylguanin-DNA-methytransferase (MGMT) promoter methylation is associated with survival in patients with glioblastoma. Current evidence suggests that further mismatch repair genes play a pivotal role in the tumor response to treatment. Candidate genes are MLH1, MSH2, and MSH6. Formerly, we found evidence of prognostic impact of MLH1 and MSH6 immunohistochemical expression in a small series of patients with initial glioblastoma. Two hundred and eleven patients were included who underwent macroscopically total removal of primary glioblastoma and at least one re-craniotomy for recurrence. Immunohistochemical staining was performed on paraffin-embedded specimens of initial tumors with specific antibodies against MLH1, MSH2, and MSH6. RESULTS were compared to the Ki67 proliferation index and patient survival. Additionally, fresh frozen samples from 16 paired initial and recurrent specimens were examined using real-time reverse transcription polymerase chain reaction (RT-PCR) with specific primers against MLH1, MSH2, and MSH6. RESULTS were compared to MGMT status and survival. (1) Immunohistochemical expression of MSH6 was significantly associated with the Ki67 proliferation index (P<0.001) but not with survival. (2) PCR revealed two patients with increasing expression of MLH1, MLH2, and MSH6 over treatment combined with lacking MGMT methylation. In another two patients, decreased MLH1, MSH2, and MSH6 expression was observed in combination with MGMT promoter methylation. Our data indicate that there may be glioblastoma patient subgroups characterized by MMR-expression changes beyond MGMT promoter methylation. The immunohistochemical expression of MLH1, MSH2, and MSH6 in initial glioblastoma is not associated with patient survival.

Gemcitabine and oxaliplatin or alkylating agents for neuroendocrine tumors: Comparison of efficacy and search for predictive factors guiding treatment choice.

PubMed

Dussol, Anne-Sophie; Joly, Marie-Odile; Vercherat, Cecile; Forestier, Julien; Hervieu, Valérie; Scoazec, Jean-Yves; Lombard-Bohas, Catherine; Walter, Thomas

2015-10-01

The alkylating agents (ALKYs) streptozotocin, dacarbazine, and temozolomide currently are the main drugs used in systemic chemotherapy for neuroendocrine tumors (NETs). The promising activity shown by gemcitabine and oxaliplatin (GEMOX) in previous studies prompted this study 1) to confirm the use of GEMOX in a larger population of NET patients, 2) to compare its efficacy with that of ALKYs, and 3) to explore whether the O(6) -methylguanine-DNA methyltransferase (MGMT) status could help in selecting the chemotherapy regimen. One hundred four patients with metastatic NETs (37 pancreatic NETs, 33 gastrointestinal NETs, 23 bronchial NETs, and 11 NETs of other/unknown origin) were treated with GEMOX between 2004 and 2014. Among these patients, 63 also received ALKYs. MGMT promoter gene methylation was assessed via pyrosequencing in 42 patients. Patients received a median of 6 courses of GEMOX. Twenty-four (23%) had an objective response (OR). The median progression-free survival (PFS) and overall survival were 7.8 and 31.6 months, respectively. In the 63 patients treated with both ALKYs and GEMOX, the ORs (22% and 22%) and the PFSs (7.5 and 7.3 months) were similar. The response was concordant in 53% of the patients. Promoter gene methylation of MGMT was associated with better outcomes with ALKYs (P = .03 for OR and P = .04 for PFS) but not GEMOX. GEMOX is effective against NETs; its activity is comparable to that of ALKYs, and it is not influenced by the MGMT status. Our data suggest that GEMOX might be preferred for patients with unmethylated MGMT tumors. Cancer 2015;121:3435-43. © 2015 American Cancer Society. © 2015 American Cancer Society.

Overcoming temozolomide resistance in glioblastoma via dual inhibition of NAD+ biosynthesis and base excision repair

PubMed Central

Goellner, Eva M.; Grimme, Bradford; Brown, Ashley R.; Lin, Ying-Chih; Wang, Xiao-Hong; Sugrue, Kelsey F.; Mitchell, Leah; Trivedi, Ram N.; Tang, Jiang-bo; Sobol, Robert W.

2011-01-01

Glioblastoma multiforme (GBM) is a devastating brain tumor with poor prognosis and low median survival time. Standard treatment includes radiation and chemotherapy with the DNA alkylating agent temozolomide (TMZ). However, a large percentage of tumors are resistant to the cytotoxic effects of the TMZ-induced DNA lesion O6-methylguanine (O6-MeG) due to elevated expression of the repair protein O6-methylguanine-DNA methyltransferase (MGMT) or a defect in the mismatch repair (MMR) pathway. Although a majority of the TMZ induced lesions (N7-methylguanine and N3-methyladenine) are base excision repair (BER) substrates, these DNA lesions are also readily repaired. However, blocking BER can enhance response to TMZ and therefore the BER pathway has emerged as an attractive target for reversing TMZ resistance. Our lab has recently reported that inhibition of BER leads to the accumulation of repair intermediates that induce energy depletion-mediated cell death via hyperactivation of poly(ADP-ribose) polymerase. Based on our observation that TMZ-induced cell death via BER inhibition is dependent on the availability of NAD+, we have hypothesized that combined BER and NAD+ biosynthesis inhibition will increase TMZ efficacy in glioblastoma cell lines greater than BER inhibition alone. Importantly, we find that the combination of BER and NAD+ biosynthesis inhibition significantly sensitizes glioma cells with elevated expression of MGMT and those deficient in MMR, two genotypes normally associated with TMZ resistance. Dual targeting of these two interacting pathways (DNA repair and NAD+ biosynthesis) may prove to be an effective treatment combination for patients with resistant and recurrent GBM. PMID:21406402

Rapid electrochemical assessment of tumor suppressor gene methylations in raw human serum, and tumor cells and tissues using immuno-magnetic beads and selective DNA hybridization.

PubMed

Povedano, Eloy; Valverde, Alejandro; Ruiz-Valdepeñas Montiel, Víctor; Pedrero, María; Yáñez-Sedeño, Paloma; Barderas, Rodrigo; San Segundo-Acosta, Pablo; Peláez-García, Alberto; Mendiola, Marta; Hardisson, David; Campuzano, Susana; Pingarron, José Manuel

2018-05-09

We report a rapid and sensitive electrochemical strategy for the detection of gene-specific 5-methylcytosine DNA methylation. Magnetic beads (MBs) modified with an antibody specific for 5-methylcytosines (5-mC) are employed for the selective capture of any 5-mC methylated single-stranded (ss)DNA sequence. A flanking region next to the 5-mCs of the captured methylated ssDNA is recognized by selective hybridization with a synthetic biotinylated DNA sequence, further labeled with an HRP streptavidin conjugate. Amperometric transduction at disposable screen-printed carbon electrodes (SPCEs) is employed. The developed biosensor exhibits a dynamic range from 3.9 to 500 pM and a detection limit of 1.2 pM for the methylated synthetic sequence of the tumor suppressor gene O-6-methylguanine-DNA methyltransferase (MGMT) promoter region. The applicability of this strategy is demonstrated through the 45 min-analysis of specific methylation in the MGMT promoter region directly in raw spiked human serum samples and in genomic DNA extracted from U-87 glioblastoma cells and paraffin-embedded brain tumor tissues without any amplification and pretreatment step. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma.

PubMed

Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

2013-12-01

Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours.

A huge intraventricular congenital anaplastic astrocytoma: case report with histopathological and genetic consideration.

PubMed

Yamashita, Shinji; Ryu, Shinitsu; Miyata, Shiro; Uchinokura, Syunrou; Yokogami, Kiyotaka; Uehara, Hisao; Moriguchi, Sayaka; Iwakiri, Takashi; Marutsuka, Kousuke; Ikenoue, Makoto; Sawa, Daisuke; Yamada, Naoshi; Kodama, Yuki; Takeshima, Hideo

2012-04-01

Congenital malignant gliomas are rare brain tumors about which few reports have been published. We present the clinical course and genetic alterations in an infant with a congenital malignant glioma detected incidentally by ultrasonography at 36 weeks. The tumor occupied the right temporoparietal region, extended to the posterior fossa, and significantly compressed surrounding structures. The female infant was entirely normal without macrocrania, tense fontanel, or sucking difficulties. The tumor was subtotally resected by two-stage surgery; pathological diagnosis was anaplastic astrocytoma. Immunohistochemical staining was positive for p53 and negative for epidermal growth factor receptor. There was no O(6)-methylguanine-DNA methyltransferase (MGMT) gene promoter methylation, no 1p/19q loss of heterozygosity, and no isocitrate dehydrogenase 1 (IDH1) mutation. She underwent postoperative chemotherapy and is alive and well 12 months after surgery.

Repair of O6-methylguanine adducts in human telomeric G-quadruplex DNA by O6-alkylguanine-DNA alkyltransferase

PubMed Central

Hellman, Lance M.; Spear, Tyler J.; Koontz, Colton J.; Melikishvili, Manana; Fried, Michael G.

2014-01-01

O6-alkylguanine-DNA alkyltransferase (AGT) is a single-cycle DNA repair enzyme that removes pro-mutagenic O6-alkylguanine adducts from DNA. Its functions with short single-stranded and duplex substrates have been characterized, but its ability to act on other DNA structures remains poorly understood. Here, we examine the functions of this enzyme on O6-methylguanine (6mG) adducts in the four-stranded structure of the human telomeric G-quadruplex. On a folded 22-nt G-quadruplex substrate, binding saturated at 2 AGT:DNA, significantly less than the ∼5 AGT:DNA found with linear single-stranded DNAs of similar length, and less than the value found with the telomere sequence under conditions that inhibit quadruplex formation (4 AGT:DNA). Despite these differences, AGT repaired 6mG adducts located within folded G-quadruplexes, at rates that were comparable to those found for a duplex DNA substrate under analogous conditions. Repair was kinetically biphasic with the amplitudes of rapid and slow phases dependent on the position of the adduct within the G-quadruplex: in general, adducts located in the top or bottom tetrads of a quadruplex stack exhibited more rapid-phase repair than did adducts located in the inner tetrad. This distinction may reflect differences in the conformational dynamics of 6mG residues in G-quadruplex DNAs. PMID:25080506

Influence of DNA repair on nonlinear dose-responses for mutation.

PubMed

Thomas, Adam D; Jenkins, Gareth J S; Kaina, Bernd; Bodger, Owen G; Tomaszowski, Karl-Heinz; Lewis, Paul D; Doak, Shareen H; Johnson, George E

2013-03-01

Recent evidence has challenged the default assumption that all DNA-reactive alkylating agents exhibit a linear dose-response. Emerging evidence suggests that the model alkylating agents methyl- and ethylmethanesulfonate and methylnitrosourea (MNU) and ethylnitrosourea observe a nonlinear dose-response with a no observed genotoxic effect level (NOGEL). Follow-up mechanistic studies are essential to understand the mechanism of cellular tolerance and biological relevance of such NOGELs. MNU is one of the most mutagenic simple alkylators. Therefore, understanding the mechanism of mutation induction, following low-dose MNU treatment, sets precedence for weaker mutagenic alkylating agents. Here, we tested MNU at 10-fold lower concentrations than a previous study and report a NOGEL of 0.0075 µg/ml (72.8nM) in human lymphoblastoid cells, quantified through the hypoxanthine (guanine) phosphoribosyltransferase assay (OECD 476). Mechanistic studies reveal that the NOGEL is dependent upon repair of O(6)-methylguanine (O(6)MeG) by the suicide enzyme O(6)MeG-DNA methyltransferase (MGMT). Inactivation of MGMT sensitizes cells to MNU-induced mutagenesis and shifts the NOGEL to the left on the dose axis.

Reduction of MLH1 and PMS2 confers temozolomide resistance and is associated with recurrence of glioblastoma

PubMed Central

Shinsato, Yoshinari; Furukawa, Tatsuhiko; Yunoue, Shunji; Yonezawa, Hajime; Minami, Kentarou; Nishizawa, Yukihiko; Ikeda, Ryuji; Kawahara, Kohichi; Yamamoto, Masatatsu; Hirano, Hirofumi; Tokimura, Hiroshi; Arita, Kazunori

2013-01-01

Although there is a relationship between DNA repair deficiency and temozolomide (TMZ) resistance in glioblastoma (GBM), it remains unclear which molecule is associated with GBM recurrence. We isolated three TMZ-resistant human GBM cell lines and examined the expression of O6-methylguanine-DNA methyltransferase (MGMT) and mismatch repair (MMR) components. We used immunohistochemical analysis to compare MutL homolog 1 (MLH1), postmeiotic segregation increased 2 (PMS2) and MGMT expression in primary and recurrent GBM specimens obtained from GBM patients during TMZ treatment. We found a reduction in MLH1 expression and a subsequent reduction in PMS2 protein levels in TMZ-resistant cells. Furthermore, MLH1 or PMS2 knockdown confered TMZ resistance. In recurrent GBM tumours, the expression of MLH1 and PMS2 was reduced when compared to primary tumours. PMID:24259277

Cytosine methylation effects on the repair of O6-methylguanines within CG dinucleotides.

PubMed

Guza, Rebecca; Ma, Linan; Fang, Qingming; Pegg, Anthony E; Tretyakova, Natalia

2009-08-21

O(6)-alkyldeoxyguanine adducts induced by tobacco-specific nitrosamines are repaired by O(6)-alkylguanine DNA alkyltransferase (AGT), which transfers the O(6)-alkyl group from the damaged base to a cysteine residue within the protein. In the present study, a mass spectrometry-based approach was used to analyze the effects of cytosine methylation on the kinetics of AGT repair of O(6)-methyldeoxyguanosine (O(6)-Me-dG) adducts placed within frequently mutated 5'-CG-3' dinucleotides of the p53 tumor suppressor gene. O(6)-Me-dG-containing DNA duplexes were incubated with human recombinant AGT protein, followed by rapid quenching, acid hydrolysis, and isotope dilution high pressure liquid chromatography-electrospray ionization tandem mass spectrometry analysis of unrepaired O(6)-methylguanine. Second-order rate constants were calculated in the absence or presence of the C-5 methyl group at neighboring cytosine residues. We found that the kinetics of AGT-mediated repair of O(6)-Me-dG were affected by neighboring 5-methylcytosine ((Me)C) in a sequence-dependent manner. AGT repair of O(6)-Me-dG adducts placed within 5'-CG-3' dinucleotides of p53 codons 245 and 248 was hindered when (Me)C was present in both DNA strands. In contrast, cytosine methylation within p53 codon 158 slightly increased the rate of O(6)-Me-dG repair by AGT. The effects of (Me)C located immediately 5' and in the base paired position to O(6)-Me-dG were not additive as revealed by experiments with hypomethylated sequences. Furthermore, differences in dealkylation rates did not correlate with AGT protein affinity for cytosine-methylated and unmethylated DNA duplexes or with the rates of AGT-mediated nucleotide flipping, suggesting that (Me)C influences other kinetic steps involved in repair, e.g. the rate of alkyl transfer from DNA to AGT.

Intratumoral heterogeneity identified at the epigenetic, genetic and transcriptional level in glioblastoma.

PubMed

Parker, Nicole R; Hudson, Amanda L; Khong, Peter; Parkinson, Jonathon F; Dwight, Trisha; Ikin, Rowan J; Zhu, Ying; Cheng, Zhangkai Jason; Vafaee, Fatemeh; Chen, Jason; Wheeler, Helen R; Howell, Viive M

2016-03-04

Heterogeneity is a hallmark of glioblastoma with intratumoral heterogeneity contributing to variability in responses and resistance to standard treatments. Promoter methylation status of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT) is the most important clinical biomarker in glioblastoma, predicting for therapeutic response. However, it does not always correlate with response. This may be due to intratumoral heterogeneity, with a single biopsy unlikely to represent the entire lesion. Aberrations in other DNA repair mechanisms may also contribute. This study investigated intratumoral heterogeneity in multiple glioblastoma tumors with a particular focus on the DNA repair pathways. Transcriptional intratumoral heterogeneity was identified in 40% of cases with variability in MGMT methylation status found in 14% of cases. As well as identifying intratumoral heterogeneity at the transcriptional and epigenetic levels, targeted next generation sequencing identified between 1 and 37 unique sequence variants per specimen. In-silico tools were then able to identify deleterious variants in both the base excision repair and the mismatch repair pathways that may contribute to therapeutic response. As these pathways have roles in temozolomide response, these findings may confound patient management and highlight the importance of assessing multiple tumor biopsies.

Reduction of Werner Syndrome Protein Enhances G:C → A:T Transition by O6-Methylguanine in Human Cells.

PubMed

Suzuki, Tetsuya; Kuramoto, Yoshie; Kamiya, Hiroyuki

2018-05-21

O 6 -Methylguanine ( O 6 -MeG) is a damaged base produced by methylating reagents. The Werner syndrome protein (WRN) is a cancer-related human DNA helicase. The effects of WRN reduction on O 6 -MeG-caused mutagenesis were assessed by an siRNA-mediated knockdown in human U2OS cells, using a shuttle plasmid with a single O 6 -MeG base in the supF gene. The plasmid DNA was replicated in the cells, isolated, and electroporated into an Escherichia coli indicator strain. The lowered amount of WRN increased the frequency of mutations induced by O 6 -MeG, mainly G:C → A:T substitution. The increased mutation rate suggested that the cancer-related WRN suppresses the G:C → A:T substitution by O 6 -MeG in human cells.

Cytosine Methylation Effects on the Repair of O6-Methylguanines within CG Dinucleotides*

PubMed Central

Guza, Rebecca; Ma, Linan; Fang, Qingming; Pegg, Anthony E.; Tretyakova, Natalia

2009-01-01

O6-Alkyldeoxyguanine adducts induced by tobacco-specific nitrosamines are repaired by O6-alkylguanine DNA alkyltransferase (AGT), which transfers the O6-alkyl group from the damaged base to a cysteine residue within the protein. In the present study, a mass spectrometry-based approach was used to analyze the effects of cytosine methylation on the kinetics of AGT repair of O6-methyldeoxyguanosine (O6-Me-dG) adducts placed within frequently mutated 5′-CG-3′ dinucleotides of the p53 tumor suppressor gene. O6-Me-dG-containing DNA duplexes were incubated with human recombinant AGT protein, followed by rapid quenching, acid hydrolysis, and isotope dilution high pressure liquid chromatography-electrospray ionization tandem mass spectrometry analysis of unrepaired O6-methylguanine. Second-order rate constants were calculated in the absence or presence of the C-5 methyl group at neighboring cytosine residues. We found that the kinetics of AGT-mediated repair of O6-Me-dG were affected by neighboring 5-methylcytosine (MeC) in a sequence-dependent manner. AGT repair of O6-Me-dG adducts placed within 5′-CG-3′ dinucleotides of p53 codons 245 and 248 was hindered when MeC was present in both DNA strands. In contrast, cytosine methylation within p53 codon 158 slightly increased the rate of O6-Me-dG repair by AGT. The effects of MeC located immediately 5′ and in the base paired position to O6-Me-dG were not additive as revealed by experiments with hypomethylated sequences. Furthermore, differences in dealkylation rates did not correlate with AGT protein affinity for cytosine-methylated and unmethylated DNA duplexes or with the rates of AGT-mediated nucleotide flipping, suggesting that MeC influences other kinetic steps involved in repair, e.g. the rate of alkyl transfer from DNA to AGT. PMID:19531487

Influence of DNA Repair on Nonlinear Dose-Responses for Mutation

PubMed Central

Johnson, George E.

2013-01-01

Recent evidence has challenged the default assumption that all DNA-reactive alkylating agents exhibit a linear dose-response. Emerging evidence suggests that the model alkylating agents methyl- and ethylmethanesulfonate and methylnitrosourea (MNU) and ethylnitrosourea observe a nonlinear dose-response with a no observed genotoxic effect level (NOGEL). Follow-up mechanistic studies are essential to understand the mechanism of cellular tolerance and biological relevance of such NOGELs. MNU is one of the most mutagenic simple alkylators. Therefore, understanding the mechanism of mutation induction, following low-dose MNU treatment, sets precedence for weaker mutagenic alkylating agents. Here, we tested MNU at 10-fold lower concentrations than a previous study and report a NOGEL of 0.0075 µg/ml (72.8nM) in human lymphoblastoid cells, quantified through the hypoxanthine (guanine) phosphoribosyltransferase assay (OECD 476). Mechanistic studies reveal that the NOGEL is dependent upon repair of O6-methylguanine (O6MeG) by the suicide enzyme O6MeG-DNA methyltransferase (MGMT). Inactivation of MGMT sensitizes cells to MNU-induced mutagenesis and shifts the NOGEL to the left on the dose axis. PMID:23288051

Concurrent Chemoradiotherapy with Temozolomide Followed by Adjuvant Temozolomide for Newly Diagnosed Glioblastoma Patients: A Retrospective Multicenter Observation Study in Korea.

PubMed

Kim, Byung Sup; Seol, Ho Jun; Nam, Do-Hyun; Park, Chul-Kee; Kim, Il Han; Kim, Tae Min; Kim, Jeong Hoon; Cho, Young Hyun; Yoon, Sang Min; Chang, Jong Hee; Kang, Seok-Gu; Kim, Eui Hyun; Suh, Chang-Ok; Jung, Tae-Young; Lee, Kyung-Hwa; Kim, Chae-Yong; Kim, In Ah; Hong, Chang-Ki; Yoo, Heon; Kim, Jin Hee; Kang, Shin-Hyuk; Kang, Min Kyu; Kim, Eun-Young; Kim, Sun-Hwan; Chung, Dong-Sup; Hwang, Sun-Chul; Song, Joon-Ho; Cho, Sung Jin; Lee, Sun-Il; Lee, Youn-Soo; Ahn, Kook-Jin; Kim, Se Hoon; Lim, Do Hun; Gwak, Ho-Shin; Lee, Se-Hoon; Hong, Yong-Kil

2017-01-01

The purpose of this study was to investigate the feasibility and survival benefits of combined treatment with radiotherapy and adjuvant temozolomide (TMZ) in a Korean sample. A total of 750 Korean patients with histologically confirmed glioblastoma multiforme, who received concurrent chemoradiotherapy with TMZ (CCRT) and adjuvant TMZ from January 2006 until June 2011, were analyzed retrospectively. After the first operation, a gross total resection (GTR), subtotal resection (STR), partial resection (PR), biopsy alone were achieved in 388 (51.7%), 159 (21.2%), 96 (12.8%), and 107 (14.3%) patients, respectively. The methylation status of O 6 -methylguanine-DNA methyltransferase (MGMT) was reviewed retrospectively in 217 patients. The median follow-up period was 16.3 months and the median overall survival (OS) was 17.5 months. The actuarial survival rates at the 1-, 3-, and 5-year OS were 72.1%, 21.0%, and 9.0%, respectively. The median progression-free survival (PFS) was 10.1 months, and the actuarial PFS at 1-, 3-, and 5-year PFS were 42.2%, 13.0%, and 7.8%, respectively. The patients who received GTR showed a significantly longer OS and PFS than those who received STR, PR, or biopsy alone, regardless of the methylation status of the MGMT promoter. Patients with a methylated MGMT promoter also showed a significantly longer OS and PFS than those with an unmethylated MGMT promoter. Patients who received more than six cycles of adjuvant TMZ had a longer OS and PFS than those who received six or fewer cycles. Hematologic toxicity of grade 3 or 4 was observed in 8.4% of patients during the CCRT period and in 10.2% during the adjuvant TMZ period. Patients treated with CCRT followed by adjuvant TMZ had more favorable survival rates and tolerable toxicity than those who did not undergo this treatment.

Clinical Neuropathology practice news 1-2014: Pyrosequencing meets clinical and analytical performance criteria for routine testing of MGMT promoter methylation status in glioblastoma

PubMed Central

Preusser, Matthias; Berghoff, Anna S.; Manzl, Claudia; Filipits, Martin; Weinhäusel, Andreas; Pulverer, Walter; Dieckmann, Karin; Widhalm, Georg; Wöhrer, Adelheid; Knosp, Engelbert; Marosi, Christine; Hainfellner, Johannes A.

2014-01-01

Testing of the MGMT promoter methylation status in glioblastoma is relevant for clinical decision making and research applications. Two recent and independent phase III therapy trials confirmed a prognostic and predictive value of the MGMT promoter methylation status in elderly glioblastoma patients. Several methods for MGMT promoter methylation testing have been proposed, but seem to be of limited test reliability. Therefore, and also due to feasibility reasons, translation of MGMT methylation testing into routine use has been protracted so far. Pyrosequencing after prior DNA bisulfite modification has emerged as a reliable, accurate, fast and easy-to-use method for MGMT promoter methylation testing in tumor tissues (including formalin-fixed and paraffin-embedded samples). We performed an intra- and inter-laboratory ring trial which demonstrates a high analytical performance of this technique. Thus, pyrosequencing-based assessment of MGMT promoter methylation status in glioblastoma meets the criteria of high analytical test performance and can be recommended for clinical application, provided that strict quality control is performed. Our article summarizes clinical indications, practical instructions and open issues for MGMT promoter methylation testing in glioblastoma using pyrosequencing. PMID:24359605

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota.

PubMed

Pence, Matthew G; Choi, Jeong-Yun; Egli, Martin; Guengerich, F Peter

2010-12-24

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

Error-prone bypass of O6-methylguanine by DNA polymerase of Pseudomonas aeruginosa phage PaP1.

PubMed

Gu, Shiling; Xiong, Jingyuan; Shi, Ying; You, Jia; Zou, Zhenyu; Liu, Xiaoying; Zhang, Huidong

2017-09-01

O 6 -Methylguanine (O 6 -MeG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, generally leads to G:C to A:T mutagenesis. To study DNA replication encountering O 6 -MeG by the DNA polymerase (gp90) of P. aeruginosa phage PaP1, we analyzed steady-state and pre-steady-state kinetics of nucleotide incorporation opposite O 6 -MeG by gp90 exo - . O 6 -MeG partially inhibited full-length extension by gp90 exo - . O 6 -MeG greatly reduces dNTP incorporation efficiency, resulting in 67-fold preferential error-prone incorporation of dTTP than dCTP. Gp90 exo - extends beyond T:O 6 -MeG 2-fold more efficiently than C:O 6 -MeG. Incorporation of dCTP opposite G and incorporation of dCTP or dTTP opposite O 6 -MeG show fast burst phases. The pre-steady-state incorporation efficiency (k pol /K d,dNTP ) is decreased in the order of dCTP:G>dTTP:O 6 -MeG>dCTP:O 6 -MeG. The presence of O 6 -MeG at template does not affect the binding affinity of polymerase to DNA but it weakened their binding in the presence of dCTP and Mg 2+ . Misincorporation of dTTP opposite O 6 -MeG further weakens the binding affinity of polymerase to DNA. The priority of dTTP incorporation opposite O 6 -MeG is originated from the fact that dTTP can induce a faster conformational change step and a faster chemical step than dCTP. This study reveals that gp90 bypasses O 6 -MeG in an error-prone manner and provides further understanding in DNA replication encountering mutagenic alkylation DNA damage for P. aeruginosa phage PaP1. Copyright © 2017 Elsevier B.V. All rights reserved.

Diversity of cytogenetic and pathohistologic profiles in glioblastoma.

PubMed

Hassler, Marco; Seidl, Sonja; Fazeny-Doerner, Barbara; Preusser, Matthias; Hainfellner, Johannes; Rössler, Karl; Prayer, Daniela; Marosi, Christine

2006-04-01

We present a small series of patients with primary glioblastoma multiforme (GBM), and combine individual genetic data with pathohistologic characteristics and clinical outcome. Eighteen patients (12 men, 6 women, median age 51 years) with histologically proven GBM underwent surgical debulking followed by radiotherapy. Fifteen received concomitant chemotherapy. Histologic typing, immunohistochemistry for CD34, karyotypic analysis, and classification of the pattern of neovascularization was done in all patients. In 12/18, we performed methylation-specific polymerase chain reaction of the MGMT gene (O-6-methylguanine-DNA methyltransferase). The survival duration of patients spanned 3-58 months. By classical banding methods, 15/18 patients showed at least one aberration characteristic for primary glioblastoma (+7 in 7/18, deletions of 9p in 10/18 and -10 or deletions from 10q in 8/18 patients). We could not assess whether patients who survived for longer periods showed less complex or fewer aberrations than the patients who survived less than one year. Losses of 6p21(VEGF), 4q27(bFGF), and 12p11 approximately p13 (ING4) were associated with the "bizarre" pattern of neoangiogenesis. Methylation of the MGMT promoter was found in 3/12 patients. Even in this small series, the main characteristic of GBM was its diversity regarding all investigated histologic and genetic characteristics. This extreme diversity should be considered in the design of targeted therapies in GBM.

In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene

PubMed Central

Larochelle, Andre; Choi, Uimook; Shou, Yan; Naumann, Nora; Loktionova, Natalia A.; Clevenger, Joshua R.; Krouse, Allen; Metzger, Mark; Donahue, Robert E.; Kang, Elizabeth; Stewart, Clinton; Persons, Derek; Malech, Harry L.; Dunbar, Cynthia E.; Sorrentino, Brian P.

2009-01-01

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen. PMID:19509470

Extracts of chronic lymphocytic leukemia lymphocytes have a high level of DNA repair activity for O/sup 6/-methylguanine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waldstein, E.A.; Cao, E.H.; Miller, M.E.

Extracts of peripheral lymphocytes from six individuals with chronic lymphocytic leukemia (CLL) were assayed for the ability to remove O/sup 6/-methylguanine (O/sup 6/MeGua) from exogenous DNA. The O/sup 6/MeGua-removing activity in CLL lymphocytes, predominantly B cells, was approximately 7-fold higher than in B lymphocytes of normal individuals and about 2-fold higher than in the unstimulated T type cells of normal persons. The activity measured in extracts of lymphocytes from three blood relatives was in the upper range of the normal distribution. Over 80% of the removal of O/sup 6/MeGua was accomplished by the transfer of the methyl group to cysteinemore » moieties of acceptor proteins in a stoichiometric reaction. If one assumes one acceptor group per acceptor protein, the calculated number of acceptor molecules per CLL lymphocyte falls between 91,000 and 220,000. Thus CLL lymphocytes do not show lower O/sup 6/MeGua-removing activity, in contrast to many tumor cell strains or transformed cell lines, which are reported to have a deficient methyl excision repair phenotype (Mer/sup -/). Instead, the CLL lymphocytes act as if they have a super-Mer/sup +/ phenotype.« less

MARCKS Regulates Growth, Radiation Sensitivity and is a Novel Prognostic Factor for Glioma

PubMed Central

Jarboe, John S.; Anderson, Joshua C.; Duarte, Christine W.; Mehta, Tapan; Nowsheen, Somaira; Hicks, Patricia H.; Whitley, Alexander C.; Rohrbach, Timothy D.; McCubrey, Raymond O.; Chiu, Sherard; Burleson, Tamara M.; Bonner, James A.; Gillespie, G. Yancey; Yang, Eddy S.; Willey, Christopher D.

2013-01-01

Purpose This study assessed whether Myristoylated Alanine Rich C-Kinase Substrate (MARCKS) can regulate glioblastoma (GBM) growth, radiation sensitivity and clinical outcome. Experimental Design MARCKS protein levels were analyzed in five GBM explant cell lines and eight patient-derived xenograft tumors by immunoblot, and these levels were correlated to proliferation rates and intracranial growth rates, respectively. Manipulation of MARCKS protein levels was assessed by lentiviral-mediated shRNA knockdown in the U251 cell line and MARCKS over-expression in the U87 cell line. The effect of manipulation of MARCKS on proliferation, radiation sensitivity and senescence was assessed. MARCKS gene expression was correlated with survival outcomes in the Repository of Molecular Brain Neoplasia Data (REMBRANDT) Database and The Cancer Genome Atlas (TCGA). Results MARCKS protein expression was inversely correlated with GBM proliferation and intracranial xenograft growth rates. Genetic silencing of MARCKS promoted GBM proliferation and radiation resistance, while MARCKS overexpression greatly reduced GBM growth potential and induced senescence. We found MARCKS gene expression to be directly correlated with survival in both the REMBRANDT and TCGA databases. Specifically, patients with high MARCKS expressing tumors of the Proneural molecular subtype had significantly increased survival rates. This effect was most pronounced in tumors with unmethylated O6-methylguanine DNA methyltransferase (MGMT) promoters, a traditionally poor prognostic factor. Conclusions MARCKS levels impact GBM growth and radiation sensitivity. High MARCKS expressing GBM tumors are associated with improved survival, particularly with unmethylated MGMT promoters. These findings suggest the use of MARCKS as a novel target and biomarker for prognosis in the Proneural subtype of GBM. PMID:22619307

DNA Damage Induced Neuronal Death

DTIC Science & Technology

1999-10-01

heterozygous for the DNA repair genes Os-methylguanine methyltransferase (Mgmt), 3-methyladenine DNA glycosylase (Aag) , and xeroderma pigmentosum ...mice by human 06-alkylguanine-DNA alkyltransferase. Science 1993; 259: 219-222. 4. Enokido Y, Inamura N, Araki T, et al: Loss of the xeroderma ... pigmentosum group A gene (XPA) enhances apoptosis of cultured cerebellar neurons induced by UV but not by low-K+ medium. J Neurochem 199; 69: 246-251. 5

Dose-dense temozolomide for newly diagnosed glioblastoma: a randomized phase III clinical trial.

PubMed

Gilbert, Mark R; Wang, Meihua; Aldape, Kenneth D; Stupp, Roger; Hegi, Monika E; Jaeckle, Kurt A; Armstrong, Terri S; Wefel, Jeffrey S; Won, Minhee; Blumenthal, Deborah T; Mahajan, Anita; Schultz, Christopher J; Erridge, Sara; Baumert, Brigitta; Hopkins, Kristen I; Tzuk-Shina, Tzahala; Brown, Paul D; Chakravarti, Arnab; Curran, Walter J; Mehta, Minesh P

2013-11-10

Radiotherapy with concomitant and adjuvant temozolomide is the standard of care for newly diagnosed glioblastoma (GBM). O(6)-methylguanine-DNA methyltransferase (MGMT) methylation status may be an important determinant of treatment response. Dose-dense (DD) temozolomide results in prolonged depletion of MGMT in blood mononuclear cells and possibly in tumor. This trial tested whether DD temozolomide improves overall survival (OS) or progression-free survival (PFS) in patients with newly diagnosed GBM. This phase III trial enrolled patients older than age 18 years with a Karnofsky performance score of ≥ 60 with adequate tissue. Stratification included clinical factors and tumor MGMT methylation status. Patients were randomly assigned to standard temozolomide (arm 1) or DD temozolomide (arm 2) for 6 to 12 cycles. The primary end point was OS. Secondary analyses evaluated the impact of MGMT status. A total of 833 patients were randomly assigned to either arm 1 or arm 2 (1,173 registered). No statistically significant difference was observed between arms for median OS (16.6 v 14.9 months, respectively; hazard ratio [HR], 1.03; P = .63) or median PFS (5.5 v 6.7 months; HR, 0.87; P = .06). Efficacy did not differ by methylation status. MGMT methylation was associated with improved OS (21.2 v 14 months; HR, 1.74; P < .001), PFS (8.7 v 5.7 months; HR, 1.63; P < .001), and response (P = .012). There was increased grade ≥ 3 toxicity in arm 2 (34% v 53%; P < .001), mostly lymphopenia and fatigue. This study did not demonstrate improved efficacy for DD temozolomide for newly diagnosed GBM, regardless of methylation status. However, it did confirm the prognostic significance of MGMT methylation. Feasibility of large-scale accrual, prospective tumor collection, and molecular stratification was demonstrated.

Dose-Dense Temozolomide for Newly Diagnosed Glioblastoma: A Randomized Phase III Clinical Trial

PubMed Central

Gilbert, Mark R.; Wang, Meihua; Aldape, Kenneth D.; Stupp, Roger; Hegi, Monika E.; Jaeckle, Kurt A.; Armstrong, Terri S.; Wefel, Jeffrey S.; Won, Minhee; Blumenthal, Deborah T.; Mahajan, Anita; Schultz, Christopher J.; Erridge, Sara; Baumert, Brigitta; Hopkins, Kristen I.; Tzuk-Shina, Tzahala; Brown, Paul D.; Chakravarti, Arnab; Curran, Walter J.; Mehta, Minesh P.

2013-01-01

Purpose Radiotherapy with concomitant and adjuvant temozolomide is the standard of care for newly diagnosed glioblastoma (GBM). O6-methylguanine-DNA methyltransferase (MGMT) methylation status may be an important determinant of treatment response. Dose-dense (DD) temozolomide results in prolonged depletion of MGMT in blood mononuclear cells and possibly in tumor. This trial tested whether DD temozolomide improves overall survival (OS) or progression-free survival (PFS) in patients with newly diagnosed GBM. Patients and Methods This phase III trial enrolled patients older than age 18 years with a Karnofsky performance score of ≥ 60 with adequate tissue. Stratification included clinical factors and tumor MGMT methylation status. Patients were randomly assigned to standard temozolomide (arm 1) or DD temozolomide (arm 2) for 6 to 12 cycles. The primary end point was OS. Secondary analyses evaluated the impact of MGMT status. Results A total of 833 patients were randomly assigned to either arm 1 or arm 2 (1,173 registered). No statistically significant difference was observed between arms for median OS (16.6 v 14.9 months, respectively; hazard ratio [HR], 1.03; P = .63) or median PFS (5.5 v 6.7 months; HR, 0.87; P = .06). Efficacy did not differ by methylation status. MGMT methylation was associated with improved OS (21.2 v 14 months; HR, 1.74; P < .001), PFS (8.7 v 5.7 months; HR, 1.63; P < .001), and response (P = .012). There was increased grade ≥ 3 toxicity in arm 2 (34% v 53%; P < .001), mostly lymphopenia and fatigue. Conclusion This study did not demonstrate improved efficacy for DD temozolomide for newly diagnosed GBM, regardless of methylation status. However, it did confirm the prognostic significance of MGMT methylation. Feasibility of large-scale accrual, prospective tumor collection, and molecular stratification was demonstrated. PMID:24101040

Phase 1 Study of Preoperative Chemoradiation Therapy With Temozolomide and Capecitabine in Patients With Locally Advanced Rectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Jae Ho; Hong, Yong Sang; Park, Yangsoon

Purpose: Preoperative chemoradiation therapy (CRT) with capecitabine is a standard treatment strategy in patients with locally advanced rectal cancer (LARC). Temozolomide improves the survival of patients with glioblastoma with hypermethylated O{sup 6}-methylguanine DNA methyltransferase (MGMT); MGMT hypermethylation is one of the colorectal carcinogenesis pathways. We aimed to determine the dose-limiting toxicity (DLT) and recommended dose (RD) of temolozomide in combination with capecitabine-based preoperative CRT for LARC. Methods and Materials: Radiation therapy was delivered with 45 Gy/25 daily fractions with coned-down boost of 5.4 Gy/3 fractions. Concurrent chemotherapy comprised fixed and escalated doses of capecitabine and temozolomide, respectively. The MGMT hypermethylation was evaluatedmore » in pretreatment tumor samples. This trial is registered with (ClinicalTrials.gov) with the number (NCT01781403). Results: Twenty-two patients with LARC of cT3-4N0 or cT{sub any}N1-2 were accrued. Dose level 3 was chosen as the RD because DLT was noticeably absent in 10 patients treated up to dose level 3. An additional 12 patients were recruited in this group. Grade III adverse events were noted, and pathologic complete response (pCR) was observed in 7 patients (31.8%); MGMT hypermethylation was detected in 16. The pCR rate was 37.5% and 16.7% in the hypermethylated and unmethylated MGMT groups, respectively (P=.616). Conclusions: There was a tendency toward higher pCR rates in patients with hypermethylated MGMT. Future randomized studies are therefore warranted.« less

Impact of DNA repair on the dose-response of colorectal cancer formation induced by dietary carcinogens.

PubMed

Fahrer, Jörg; Kaina, Bernd

2017-08-01

Colorectal cancer (CRC) is one of the most frequently diagnosed cancers, which is causally linked to dietary habits, notably the intake of processed and red meat. Processed and red meat contain dietary carcinogens, including heterocyclic aromatic amines (HCAs) and N-nitroso compounds (NOC). NOC are agents that induce various N-methylated DNA adducts and O 6 -methylguanine (O 6 -MeG), which are removed by base excision repair (BER) and O 6 -methylguanine-DNA methyltransferase (MGMT), respectively. HCAs such as the highly mutagenic 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) cause bulky DNA adducts, which are removed from DNA by nucleotide excision repair (NER). Both O 6 -MeG and HCA-induced DNA adducts are linked to the occurrence of KRAS and APC mutations in colorectal tumors of rodents and humans, thereby driving CRC initiation and progression. In this review, we focus on DNA repair pathways removing DNA lesions induced by NOC and HCA and assess their role in protecting against mutagenicity and carcinogenicity in the large intestine. We further discuss the impact of DNA repair on the dose-response relationship in colorectal carcinogenesis in view of recent studies, demonstrating the existence of 'no effect' point of departures (PoDs), i.e. thresholds for genotoxicity and carcinogenicity. The available data support the threshold concept for NOC with DNA repair being causally involved. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Nanoparticle Carrying the p53 Gene Targets Tumors Including Cancer Stem Cells, Sensitizes Glioblastoma to Chemotherapy and Improves Survival

PubMed Central

2015-01-01

Temozolomide (TMZ)-resistance in glioblastoma multiforme (GBM) has been linked to upregulation of O6-methylguanine-DNA methyltransferase (MGMT). Wild-type (wt) p53 was previously shown to down-modulate MGMT. However, p53 therapy for GBM is limited by lack of efficient delivery across the blood brain barrier (BBB). We have developed a systemic nanodelivery platform (scL) for tumor-specific targeting (primary and metastatic), which is currently in multiple clinical trials. This self-assembling nanocomplex is formed by simple mixing of the components in a defined order and a specific ratio. Here, we demonstrate that scL crosses the BBB and efficiently targets GBM, as well as cancer stem cells (CSCs), which have been implicated in recurrence and treatment resistance in many human cancers. Moreover, systemic delivery of scL-p53 down-modulates MGMT and induces apoptosis in intracranial GBM xenografts. The combination of scL-p53 and TMZ increased the antitumor efficacy of TMZ with enhanced survival benefit in a mouse model of highly TMZ-resistant GBM. scL-p53 also sensitized both CSCs and bulk tumor cells to TMZ, increasing apoptosis. These results suggest that combining scL-p53 with standard TMZ treatment could be a more effective therapy for GBM. PMID:24811110

Chloroethylating nitrosoureas in cancer therapy: DNA damage, repair and cell death signaling.

PubMed

Nikolova, Teodora; Roos, Wynand P; Krämer, Oliver H; Strik, Herwig M; Kaina, Bernd

2017-08-01

Chloroethylating nitrosoureas (CNU), such as lomustine, nimustine, semustine, carmustine and fotemustine are used for the treatment of malignant gliomas, brain metastases of different origin, melanomas and Hodgkin disease. They alkylate the DNA bases and give rise to the formation of monoadducts and subsequently interstrand crosslinks (ICL). ICL are critical cytotoxic DNA lesions that link the DNA strands covalently and block DNA replication and transcription. As a result, S phase progression is inhibited and cells are triggered to undergo apoptosis and necrosis, which both contribute to the effectiveness of CNU-based cancer therapy. However, tumor cells resist chemotherapy through the repair of CNU-induced DNA damage. The suicide enzyme O 6 -methylguanine-DNA methyltransferase (MGMT) removes the precursor DNA lesion O 6 -chloroethylguanine prior to its conversion into ICL. In cells lacking MGMT, the formed ICL evoke complex enzymatic networks to accomplish their removal. Here we discuss the mechanism of ICL repair as a survival strategy of healthy and cancer cells and DNA damage signaling as a mechanism contributing to CNU-induced cell death. We also discuss therapeutic implications and strategies based on sequential and simultaneous treatment with CNU and the methylating drug temozolomide. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural Basis for Proficient Incorporation of dTTP Opposite O[superscript 6]-Methylguanine by Human DNA Polymerase [iota

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pence, Matthew G.; Choi, Jeong-Yun; Egli, Martin

2012-03-15

O{sup 6}-Methylguanine (O{sup 6}-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase L core enzyme was determined for nucleoside triphosphate incorporation opposite O{sup 6}-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol L, which showed that dTTP incorporation occurs with high efficiency opposite O{sup 6}-methylG. Misincorporation of dTTP opposite O{sup 6}-methylG occurred with {approx}6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol L with O{sup 6}-methylG asmore » the template base and incoming dCTP or dTTP were solved and showed that O{sup 6}-methylG is rotated into the syn conformation in the pol L active site and that dTTP misincorporation by pol L is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O{sup 6}-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O{sup 6}-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O{sup 6} atoms of O{sup 6}-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O{sup 6}-methylG by human pol L, in contrast to the mispairing modes observed previously for O{sup 6}-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.« less

Promoter methylation and expression of DNA repair genes MGMT and ERCC1 in tissue and blood of rectal cancer patients.

PubMed

Shalaby, Sally M; El-Shal, Amal S; Abdelaziz, Lobna A; Abd-Elbary, Eman; Khairy, Mostafa M

2018-02-20

Rectal cancer involves one-third of colorectal cancers (CRCs). Recently, data supported that DNA methylation have a role in CRC pathogenesis. In the present study we aimed to analyze the methylation status of MGMT and ERCC1 promoter regions in blood and tissue of patients with benign and malignant rectal tumors. We also studied the methylated MGMT and ERCC1 genes and their relations with clinicopathological features. Furthermore, we suggested that methylation may play a critical function in the regulation of MGMT and ERCC1 expression. Fifty patients with non-metastatic cancer rectum and 43 patients with benign rectal lesions were involved in the study. DNA extraction from blood and rectal specimens was done to analyze the methylation status of MGMT and ERCC1 genes by methylation-specific PCR method. RNA was extracted also to determine the expression levels of these genes by real time-PCR. The frequency of MGMT and ERCC1 methylation was significantly higher in rectum cancers than in benign tumors both for the tissue and the blood (p<0.001). There was no relation between MGMT or ERCC1 methylation and clinicopathological features; while they were correlated with the response to therapy. An interesting finding that the agreement of the methylation levels in the blood and rectal tissue was classified as good (κ=0.78) for MGMT gene and as very good (κ=0.85) for ERCC1. Lastly, the MGMT and ERCC1 genes methylation was associated with down-regulation of their mRNA expression when compared with the non-methylated status. Our findings provided evidence that both blood and tumor tissue MGMT and ERCC1 methylation were associated with cancer rectum. MGMT or ERCC1 methylation in blood could be suitable non-invasive biomarkers differentiating benign and malignant rectal tumors. Furthermore, the methylation of the MGMT and ERCC1 promoter regions was associated with down-regulation of their mRNA expression. Copyright © 2017 Elsevier B.V. All rights reserved.

Promoter methylation, mRNA expression of goat tumor‑associated genes and mRNA expression of DNA methyltransferase in enzootic nasal tumors.

PubMed

Quan, Zifang; Ye, Ni; Hao, Zhongxiang; Wen, Caifang; Liao, Hong; Zhang, Manli; Luo, Lu; Cao, Sanjie; Wen, Xintian; Wu, Rui; Yan, Qigui

2015-10-01

The aim of the present study was to investigate the promoter methylation status and mRNA expression of goat tumor‑associated genes, in addition to the mRNA expression of DNA methyltransferase genes in enzootic nasal tumors (ENT). Methylation‑specific polymerase chain reaction and SYBR Green reverse transcription‑quantitative polymerase chain reaction were used to detect the methylation status and the mRNA expression levels of DNA methyltransferases (DNMTs), O6‑methylguanine‑DNA methyltransferase (MGMT), the tumor suppressor genes P73, P53, GADD45G, CHFR and THBS1, the transcription factor CEBPA, the proto‑oncogenes KRAS, NRAS and C‑myc and EGFR in 24 nasal tumor tissue samples and 20 normal nasal epithelia tissue samples. The associations between promoter methylation and DNMT, and promoter methylation and mRNA expression of the genes were analyzed. The results indicated that the expression levels of DNMT1 increased by 56% compared with those in normal nasal epithelial tissues, while MGMT, DNMT3a and DNMT3b had similar expression levels in the two tissue types. The expression levels of P53 decreased by 36.8% and those of THBS1 by 43%, while C‑myc increased by 2.9‑fold and CEBPA by 2‑fold compared with that in normal nasal epithelial tissues. GADD45G, P73, CHFR and NRAS were observed to have similar expression levels in the two tissue types. However, no expression was observed for EGFR and KRAS. CHFR, GADD45G and THBS1 were identified to be methylated in tumor suppressor genes. The methylation expression rate of the CHFR gene was ~60% in the two tissue types and for THBS1 it was 100% in the nasal tumor tissues as opposed to 20% in the normal nasal epithelial tissues. The exhaustive methylation expression rate of GADD45G was 62.5% and the partial methylation expression rate was 37.5% in nasal tumor tissue, while no methylation was observed in normal nasal epithelial tissues. C‑myc was the only gene identified to be methylated amongst proto�

Pharmacological inhibition of poly(ADP-ribose) polymerase-1 modulates resistance of human glioblastoma stem cells to temozolomide

PubMed Central

2014-01-01

Background Chemoresistance of glioblastoma multiforme (GBM) has been attributed to the presence within the tumor of cancer stem cells (GSCs). The standard therapy for GBM consists of surgery followed by radiotherapy and the chemotherapeutic agent temozolomide (TMZ). However, TMZ efficacy is limited by O6-methylguanine-DNA-methyltransferase (MGMT) and Mismatch Repair (MMR) functions. Strategies to counteract TMZ resistance include its combination with poly(ADP-ribose) polymerase inhibitors (PARPi), which hamper the repair of N-methylpurines. PARPi are also investigated as monotherapy for tumors with deficiency of homologous recombination (HR). We have investigated whether PARPi may restore GSC sensitivity to TMZ or may be effective as monotherapy. Methods Ten human GSC lines were assayed for MMR proteins, MGMT and PARP-1 expression/activity, MGMT promoter methylation and sensitivity to TMZ or PARPi, alone and in combination. Since PTEN defects are frequently detected in GBM and may cause HR dysfunction, PTEN expression was also analyzed. The statistical analysis of the differences in drug sensitivity among the cell lines was performed using the ANOVA and Bonferroni’s post-test or the non-parametric Kruskal-Wallis analysis and Dunn’s post-test for multiple comparisons. Synergism between TMZ and PARPi was analyzed by the median-effect method of Chou and Talalay. Correlation analyses were done using the Spearman’s rank test. Results All GSCs were MMR-proficient and resistance to TMZ was mainly associated with high MGMT activity or low proliferation rate. MGMT promoter hypermethylation of GSCs correlated both with low MGMT activity/expression (Spearman’s test, P = 0.004 and P = 0.01) and with longer overall survival of GBM patients (P = 0.02). Sensitivity of each GSC line to PARPi as single agent did not correlate with PARP-1 or PTEN expression. Notably, PARPi and TMZ combination exerted synergistic antitumor effects in eight out of ten GSC lines and

Downregulation of HIF-1a sensitizes U251 glioma cells to the temozolomide (TMZ) treatment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tang, Jun-Hai; Ma, Zhi-Xiong; Huang, Guo-Hao

Purpose: The aim of this study was to investigate the effect of downregulation of HIF-1α gene on human U251 glioma cells and examine the consequent changes of TMZ induced effects and explore the molecular mechanisms. Methods: U251 cell line stably expressing HIF-1α shRNA was acquired via lentiviral vector transfection. The mRNA and protein expression alterations of genes involved in our study were determined respectively by qRT-PCR and Western blot. Cell proliferation was measured by MTT assay and colony formation assay, cell invasion/migration capacity was determined by transwell invasion assay/wound healing assay, and cell apoptosis was detected by flow cytometry. Results:more » We successfully established a U251 cell line with highly efficient HIF-1α knockdown. HIF-1a downregulation sensitized U251 cells to TMZ treatment and enhanced the proliferation-inhibiting, invasion/migration-suppressing, apoptosis-inducing and differentiation-promoting effects exerted by TMZ. The related molecular mechanisms demonstrated that expression of O{sup 6}-methylguanine DNA methyltransferase gene (MGMT) and genes of Notch1 pathway were significantly upregulated by TMZ treatment. However, this upregulation was abrogated by HIF-1α knockdown. We further confirmed important regulatory roles of HIF-1α in the expression of MGMT and activation of Notch1 pathways. Conclusion: HIF-1α downregulation sensitizes U251 glioma cells to the temozolomide treatment via inhibiting MGMT expression and Notch1 pathway activation. - Highlights: • TMZ caused more significant proliferation inhibition and apoptosis in U251 cells after downregulating HIF-1α. • Under TMZ treatment, HIF-1 downregulated U251 cells exhibited weaker mobility and more differentiated state. • TMZ caused MGMT over-expression and Notch1 pathway activation, which could be abrogated by HIF-1α downregulation.« less

TRIM24 promotes glioma progression and enhances chemoresistance through activation of the PI3K/Akt signaling pathway.

PubMed

Zhang, L-H; Yin, A-A; Cheng, J-X; Huang, H-Y; Li, X-M; Zhang, Y-Q; Han, N; Zhang, X

2015-01-29

The tripartite motif protein TRIM24 (tripartite motif-containing 24) has been found to play distinct roles in tumor development and progression, according to different tumor contexts. However, it remains elusive whether TRIM24 plays a role in malignant gliomas that are the most common and deadly primary brain tumors in adults. We report here that TRIM24 expression is positively correlated with glioma malignancy and is negatively associated with prognosis of patients with newly diagnosed glioblastoma, which is the most malignant form of gliomas but displays highly heterogeneous clinical outcome. The multivariate Cox regression analysis demonstrates the independent predictive value of TRIM24 expression level for overall and progression-free survival. Knockdown of TRIM24 suppresses cell proliferation, cell cycle progression, clone formation and in vivo tumor development, whereas overexpression of TRIM24 promotes cell growth. Chromatin immunoprecipitation, real-time reverse transcription-PCR and mutation analyses demonstrate that TRIM24 binds to the PIK3CA promoter via its PHD-Bromo domain to activate the transcription of PIK3CA gene, thus enhancing phosphatidylinositide 3-kinase (PI3K)/Akt signaling. The pan-PI3K inhibitor LY294002 and small interfering RNA targeting PIK3CA both abrogate the growth-promoting effect of TRIM24. Moreover, TRIM24 regulates the expression of DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) through PI3K/Akt/nuclear factor-κB signaling transduction and enhances resistance to temozolomide, the standard chemotherapeutic agent for glioblastoma. Finally, glioblastoma patients with low TRIM24 expression benefit from chemotherapy, whereas those with high TRIM24 expression do not have such benefit. Our results suggest that TRIM24 might serve as a potential prognostic marker and therapeutic target for the management of malignant gliomas.

Ras regulation of DNA-methylation and cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patra, Samir Kumar

2008-04-01

Genome wide hypomethylation and regional hypermethylation of cancer cells and tissues remain a paradox, though it has received a convincing confirmation that epigenetic switching systems, including DNA-methylation represent a fundamental regulatory mechanism that has an impact on genome maintenance and gene transcription. Methylated cytosine residues of vertebrate DNA are transmitted by clonal inheritance through the strong preference of DNA methyltransferase, DNMT1, for hemimethylated-DNA. Maintenance of methylation patterns is necessary for normal development of mice, and aberrant methylation patterns are associated with many human tumours. DNMT1 interacts with many proteins during cell cycle progression, including PCNA, p53, EZH2 and HP1. Rasmore » family of GTPases promotes cell proliferation by its oncogenic nature, which transmits signals by multiple pathways in both lipid raft dependent and independent fashion. DNA-methylation-mediated repression of DNA-repair protein O6-methylguanine DNA methyltransferase (MGMT) gene and increased rate of K-Ras mutation at codon for amino acids 12 and 13 have been correlated with a secondary role for Ras-effector homologues (RASSFs) in tumourigenesis. Lines of evidence suggest that DNA-methylation associated repression of tumour suppressors and apoptotic genes and ceaseless proliferation of tumour cells are regulated in part by Ras-signaling. Control of Ras GTPase signaling might reduce the aberrant methylation and accordingly may reduce the risk of cancer development.« less

Low-dose fotemustine for recurrent malignant glioma: a multicenter phase II study.

PubMed

Fabi, Alessandra; Metro, Giulio; Vidiri, Antonello; Lanzetta, Gaetano; Carosi, Mariantonia; Telera, Stefano; Maschio, Marta; Russillo, Michelangelo; Sperduti, Isabella; Carapella, Carmine M; Cognetti, Francesco; Pace, Andrea

2010-11-01

Fotemustine at the conventional dose of 100 mg/m(2) is an active treatment for recurrent malignant gliomas (RMGs). However, it is associated with a relevant incidence of severe myelotoxicity, which is not justified in the palliative setting of this disease. This study was conducted to address whether administration of fotemustine at 60 mg/m(2) (induction) followed by 75 mg/m(2) (maintenance) would preserve clinical activity with the advantage of improved tolerance. Forty patients with RMGs pretreated with ≤2 lines of chemotherapy were enrolled. Median age was 57 years (26-80) and median Karnofsky performance status was 80 (60-100). Thirty-one patients (77.5%) had tissue available for analysis of the O(6)-methylguanine methyltransferase (MGMT) gene promoter which was found to be methylated in 14 cases (45%). Overall, 8 partial responses (20%) and 13 disease stabilizations (32.5%) were observed for a disease-control rate of 52.5%. At 6 months, 21% of patients were free from progression. Grades 3 and 4 platelet and white blood cell toxicity occurred in ≤10% of patients, and no patients discontinued treatment because of toxicity. No significant difference was observed for disease control rate between methylated and unmethylated patients, although a trend toward improved progression-free survival was reported for methylated patients. Low-dose fotemustine has activity comparable with that of the full-dose regimen, therefore it should be preferred for its greater tolerability. The role of MGMT gene promoter methylation status in relation to sensitivity to fotemustine is still unclear and needs further evaluation in future clinical trials.

Detecting methylation patterns of p16, MGMT, DAPK and E-cadherin genes in multiple myeloma patients.

PubMed

Yuregir, O Ozalp; Yurtcu, E; Kizilkilic, E; Kocer, N E; Ozdogu, H; Sahin, F I

2010-04-01

Multiple myeloma (MM) is a B-cell neoplasia characterized by the clonal proliferation of plasma cells. Besides known genetic abnormalities, epigenetic changes are also known to effect MM pathogenesis. DNA methylation is an epigenetic mechanism that silences genes by adding methyl groups to cytosine-guanine dinucleotides at the promoter regions. In this study, the methylation status of four genes; p16, O6-methyl guanine DNA methyl transferase (MGMT), death-associated protein kinase (DAPK) and E-cadherin (ECAD); at the time of diagnosis was investigated using methylation-specific polymerase chain reaction (MS-PCR). In the 20 cases studied; methylation of the promoter regions of p16, MGMT, DAPK and ECAD genes was detected in 10%, 40%, 10% and 45% of the cases, respectively. In 65% (13/20) of cases, at least one of the genes studied had promoter methylation; while 35% of cases (7/20) had methylated promoters of more than one gene. There was a significant correlation between promoter hypermethylation of MGMT and the presence of extramedullary involvement; but for the other genes no correlation was found regarding disease properties like age, disease stage, clinical course and the presence of lytic bone lesions. Determining the methylation profiles of genes in MM, could lead to a new understanding of the disease pathogenesis and guide the assessment of treatment options.

Aberrant DNA methylation of tumor-related genes in oral rinse: a noninvasive method for detection of oral squamous cell carcinoma.

PubMed

Nagata, Satoshi; Hamada, Tomofumi; Yamada, Norishige; Yokoyama, Seiya; Kitamoto, Sho; Kanmura, Yuji; Nomura, Masahiro; Kamikawa, Yoshiaki; Yonezawa, Suguru; Sugihara, Kazumasa

2012-09-01

The early detection of oral squamous cell carcinoma (OSCC) is important, and a screening test with high sensitivity and specificity is urgently needed. Therefore, in this study, the authors investigated the methylation status of tumor-related genes with the objective of establishing a noninvasive method for the detection of OSCC. Oral rinse samples were obtained from 34 patients with OSCC and from 24 healthy individuals (controls). The methylation status of 13 genes was determined by using methylation-specific polymerase chain reaction analysis and was quantified using a microchip electrophoresis system. Promoter methylation in each participant was screened by receiver operating characteristic analysis, and the utility of each gene's methylation status, alone and in combination with other genes, was evaluated as a tool for oral cancer detection. Eight of the 13 genes had significantly higher levels of DNA methylation in samples from patients with OSCC than in controls. The genes E-cadherin (ECAD), transmembrane protein with epidermal growth factor-like and 2 follistatin-like domains 2 (TMEFF2), retinoic acid receptor beta (RARβ), and O-6 methylguanine DNA methyltransferase (MGMT) had high sensitivity (>75%) and specificity for the detection of oral cancer. OSCC was detected with 100% sensitivity and 87.5% specificity using a combination of ECAD, TMEFF2, RARβ, and MGMT and with 97.1% sensitivity and 91.7% specificity using a combination of ECAD, TMEFF2, and MGMT. The aberrant methylation of a combination of marker genes present in oral rinse samples was used to detect OSCC with >90% sensitivity and specificity. The detection of methylated marker genes from oral rinse samples has great potential for the noninvasive detection of OSCC. Copyright © 2012 American Cancer Society.

Gene therapy enhances chemotherapy tolerance and efficacy in glioblastoma patients.

PubMed

Adair, Jennifer E; Johnston, Sandra K; Mrugala, Maciej M; Beard, Brian C; Guyman, Laura A; Baldock, Anne L; Bridge, Carly A; Hawkins-Daarud, Andrea; Gori, Jennifer L; Born, Donald E; Gonzalez-Cuyar, Luis F; Silbergeld, Daniel L; Rockne, Russell C; Storer, Barry E; Rockhill, Jason K; Swanson, Kristin R; Kiem, Hans-Peter

2014-09-01

Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT(hi)). Coadministration of O6-benzylguanine (O6BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O6BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome. We enrolled 7 newly diagnosed glioblastoma patients with MGMT(hi) tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O6BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated. Gene therapy permitted a significant increase in the mean number of tolerated O6BG/TMZ cycles (4.4 cycles per patient, P < 0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5-57+) months and OS of 20 (range 13-57+) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O6BG. These data support further development of chemoprotective gene therapy in combination with O6BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT. Clinicaltrials.gov NCT00669669. R01CA114218, R

MGMT and CALCA promoter methylation are associated with poor prognosis in testicular germ cell tumor patients

PubMed Central

Martinelli, Camila Maria da Silva; Lengert, André van Helvoort; Cárcano, Flavio Mavignier; Silva, Eduardo Caetano Albino; Brait, Mariana; Lopes, Luiz Fernando; Vidal, Daniel Onofre

2017-01-01

Testicular germ cell tumors (TGCT) represent the second main cause of cancer-related death in young men. Despite high cure rates, refractory disease results in poor prognosis. Epigenetic reprogramming occurs during the development of seminomas and non-seminomas. Understanding the molecular and genetic basis of these tumors would represent an important advance in the search for new TGCT molecular markers. Hence the frequency of methylation of a gene panel (VGF, MGMT, ADAMTS1, CALCA, HOXA9, CDKN2B, CDO1 and NANOG) was evaluated in 72 primary TGCT by quantitative methylation specific PCR. A high frequency of MGMT (90.9%, 20/22; p=0.019) and CALCA (90.5%, 19/21; p<0.026) methylation was associated with non-seminomatous tumors while CALCA methylation was also associated with refractory disease (47.4%, 09/19; p=0.005). Moreover, promoter methylation of both genes predicts poor clinical outcome for TGCT patients (5-year EFS: 50.5% vs 77.1%; p=0.032 for MGMT and 51.3% vs 77.0%; p=0.029 for CALCA). The findings of this study indicate that methylation of MGMT and CALCA are frequent and could be used as new molecular markers of prognosis in TGCT. PMID:28881587

Survival and Death Strategies in Glioma Cells: Autophagy, Senescence and Apoptosis Triggered by a Single Type of Temozolomide-Induced DNA Damage

PubMed Central

Knizhnik, Anna V.; Roos, Wynand P.; Nikolova, Teodora; Quiros, Steve; Tomaszowski, Karl-Heinz; Christmann, Markus; Kaina, Bernd

2013-01-01

Apoptosis, autophagy, necrosis and cellular senescence are key responses of cells that were exposed to genotoxicants. The types of DNA damage triggering these responses and their interrelationship are largely unknown. Here we studied these responses in glioma cells treated with the methylating agent temozolomide (TMZ), which is a first-line chemotherapeutic for this malignancy. We show that upon TMZ treatment cells undergo autophagy, senescence and apoptosis in a specific time-dependent manner. Necrosis was only marginally induced. All these effects were completely abrogated in isogenic glioma cells expressing O6-methylguanine-DNA methyltransferase (MGMT), indicating that a single type of DNA lesion, O6-methylguanine (O6MeG), is able to trigger all these responses. Studies with mismatch repair mutants and MSH6, Rad51 and ATM knockdowns revealed that autophagy induced by O6MeG requires mismatch repair and ATM, and is counteracted by homologous recombination. We further show that autophagy, which precedes apoptosis, is a survival mechanism as its inhibition greatly ameliorated the level of apoptosis following TMZ at therapeutically relevant doses (<100 µM). Cellular senescence increases with post-exposure time and, similar to autophagy, precedes apoptosis. If autophagy was abrogated, TMZ-induced senescence was reduced. Therefore, we propose that autophagy triggered by O6MeG adducts is a survival mechanism that stimulates cells to undergo senescence rather than apoptosis. Overall, the data revealed that a specific DNA adduct, O6MeG, has the capability of triggering autophagy, senescence and apoptosis and that the decision between survival and death is determined by the balance of players involved. The data also suggests that inhibition of autophagy may ameliorate the therapeutic outcome of TMZ-based cancer therapy. PMID:23383259

DNA Repair Modulates The Vulnerability of The Developing Brain to Alkylating Agents

PubMed Central

Kisby, G.E.; Olivas, A.; Park, T.; Churchwell, M.; Doerge, D.; Samson, L. D.; Gerson, S.L.; Turker, M.S.

2009-01-01

Neurons of the developing brain are especially vulnerable to environmental agents that damage DNA (i.e., genotoxicants), but the mechanism is poorly understood. The focus of the present study is to demonstrate that DNA damage plays a key role in disrupting neurodevelopment. To examine this hypothesis, we compared the cytotoxic and DNA damaging properties of the methylating agents methylazoxymethanol (MAM) and dimethyl sulfate (DMS) and the mono- and bifunctional alkylating agents chloroethylamine (CEA) and nitrogen mustard (HN2), in granule cell neurons derived from the cerebellum of neonatal wild type mice and three transgenic DNA repair strains. Wild type cerebellar neurons were significantly more sensitive to the alkylating agents DMS and HN2 than neuronal cultures treated with MAM or the half-mustard CEA. Parallel studies with neuronal cultures from mice deficient in alkylguanine DNA glycosylase (Aag-/-) or O6-methylguanine methyltransferase (Mgmt-/-), revealed significant differences in the sensitivity of neurons to all four genotoxicants. Mgmt-/- neurons were more sensitive to MAM and HN2 than the other genotoxicants and wild type neurons treated with either alkylating agent. In contrast, Aag-/- neurons were for the most part significantly less sensitive than wild type or Mgmt-/- neurons to MAM and HN2. Aag-/- neurons were also significantly less sensitive than wild type neurons treated with either DMS or CEA. Granule cell development and motor function were also more severely disturbed by MAM and HN2 in Mgmt-/- mice than in comparably treated wild type mice. In contrast, cerebellar development and motor function were well preserved in MAM treated Aag-/- or MGMT overexpressing (MgmtTg+) mice, even as compared with wild type mice suggesting that AAG protein increases MAM toxicity, whereas MGMT protein decreases toxicity. Surprisingly, neuronal development and motor function were severely disturbed in MgmtTg+ mice treated with HN2. Collectively, these in vitro

The Process and Regulatory Components of Inflammation in Brain Oncogenesis

PubMed Central

Mostofa, A.G.M.; Punganuru, Surendra R.; Madala, Hanumantha Rao; Al-Obaide, Mohammad; Srivenugopal, Kalkunte S.

2017-01-01

Central nervous system tumors comprising the primary cancers and brain metastases remain the most lethal neoplasms and challenging to treat. Substantial evidence points to a paramount role for inflammation in the pathology leading to gliomagenesis, malignant progression and tumor aggressiveness in the central nervous system (CNS) microenvironment. This review summarizes the salient contributions of oxidative stress, interleukins, tumor necrosis factor-α(TNF-α), cyclooxygenases, and transcription factors such as signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) and the associated cross-talks to the inflammatory signaling in CNS cancers. The roles of reactive astrocytes, tumor associated microglia and macrophages, metabolic alterations, microsatellite instability, O6-methylguanine DNA methyltransferase (MGMT) DNA repair and epigenetic alterations mediated by the isocitrate dehydrogenase 1 (IDH1) mutations have been discussed. The inflammatory pathways with relevance to the brain cancer treatments have been highlighted. PMID:28346397

A practical review of prognostic correlations of molecular biomarkers in glioblastoma.

PubMed

Karsy, Michael; Neil, Jayson A; Guan, Jian; Mahan, Mark A; Mark, Mahan A; Colman, Howard; Jensen, Randy L

2015-03-01

Despite extensive efforts in research and therapeutics, achieving longer survival for patients with glioblastoma (GBM) remains a formidable challenge. Furthermore, because of rapid advances in the scientific understanding of GBM, communication with patients regarding the explanations and implications of genetic and molecular markers can be difficult. Understanding the important biomarkers that play a role in GBM pathogenesis may also help clinicians in educating patients about prognosis, potential clinical trials, and monitoring response to treatments. This article aims to provide an up-to-date review that can be discussed with patients regarding common molecular markers, namely O-6-methylguanine-DNA methyltransferase (MGMT), isocitrate dehydrogenase 1 and 2 (IDH1/2), p53, epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDGFR), phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), and 1p/19q. The importance of the distinction between a prognostic and a predictive biomarker as well as clinical trials regarding these markers and their relevance to clinical practice are discussed.

The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention.

PubMed

Pendleton, Kathryn E; Chen, Beibei; Liu, Kuanqing; Hunter, Olga V; Xie, Yang; Tu, Benjamin P; Conrad, Nicholas K

2017-05-18

Maintenance of proper levels of the methyl donor S-adenosylmethionine (SAM) is critical for a wide variety of biological processes. We demonstrate that the N 6 -adenosine methyltransferase METTL16 regulates expression of human MAT2A, which encodes the SAM synthetase expressed in most cells. Upon SAM depletion by methionine starvation, cells induce MAT2A expression by enhanced splicing of a retained intron. Induction requires METTL16 and its methylation substrate, a vertebrate conserved hairpin (hp1) in the MAT2A 3' UTR. Increasing METTL16 occupancy on the MAT2A 3' UTR is sufficient to induce efficient splicing. We propose that, under SAM-limiting conditions, METTL16 occupancy on hp1 increases due to inefficient enzymatic turnover, which promotes MAT2A splicing. We further show that METTL16 is the long-unknown methyltransferase for the U6 spliceosomal small nuclear RNA (snRNA). These observations suggest that the conserved U6 snRNA methyltransferase evolved an additional function in vertebrates to regulate SAM homeostasis. Copyright © 2017 Elsevier Inc. All rights reserved.

Clinicopathological factors predictive of postoperative seizures in patients with gliomas.

PubMed

Yang, Pei; Liang, Tingyu; Zhang, Chuanbao; Cai, Jinquan; Zhang, Wei; Chen, Baoshi; Qiu, Xiaoguang; Yao, Kun; Li, Guilin; Wang, Haoyuan; Jiang, Chuanlu; You, Gan; Jiang, Tao

2016-02-01

Epilepsy is one of the most common manifestations in gliomas and has a severe effect on the life expectancy and quality of life of patients. The aim of our study was to assess the potential connections between clinicopathological factors and postoperative seizure. We retrospectively investigated a group of 147 Chinese high-grade glioma (HGG) patients with preoperative seizure to examine the correlation between postoperative seizure and clinicopathological factors and prognosis. Univariate analyses and multivariate logistic regression analyses were performed to identify factors associated with postoperative seizures. Survival function curves were calculated using the Kaplan-Meier method. 53 patients (36%) were completely seizure-free (Engel class I), and 94 (64%) experienced a postoperative seizure (Engel classes II, III, and IV). A Chi-squared analysis showed that anaplastic oligodendroglioma/anaplastic oligoastrocytoma (AO/AOA) (P=0.05), epidermal growth factor receptor (EGFR) expression (P=0.0004), O(6)-methylguanine DNA methyltransferase (MGMT) expression (P=0.011), and phosphatase and tensin homolog (PTEN) expression (P=0.045) were all significantly different. A logistic regression analysis showed that MGMT expression (P=0.05), EGFR expression (P=0.001), and AO/AOA (P=0.038) are independent factors of postoperative seizure. Patients with lower MGMT and EGFR expression and AO/AOA showed more frequent instances of postoperative seizure. Postoperative seizure showed no statistical significance on overall survival (OS) and progression-free survival (PFS). Our study identified clinicopathological factors related to postoperative seizure in HGGs and found two predictive biomarkers of postoperative seizure: MGMT and EGFR. These findings provided insight treatment strategies aimed at prolonging survival and improving quality of life. Copyright © 2016 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.

MGMT prognostic impact on glioblastoma is dependent on therapeutic modalities.

PubMed

Crinière, Emmanuelle; Kaloshi, Gentian; Laigle-Donadey, Florence; Lejeune, Julie; Auger, Nathalie; Benouaich-Amiel, Alexandra; Everhard, Sibille; Mokhtari, Karima; Polivka, Marc; Delattre, Jean-Yves; Hoang-Xuan, Khê; Thillet, Joëlle; Sanson, Marc

2007-06-01

MGMT promoter methylation, which has been correlated with the response to alkylating agents, was investigated in a retrospective series of 219 glioblastomas (GBMs) treated with various modalities. MGMT methylation had no impact on survival for the whole group, but showed a significant advantage (17.1 months vs. 13.1) for patients treated with RT+ adjuvant chemotherapy (relative risk of death (RR) = 0.53; P = 0.041), particularly when patients received CT during the course of RT (MS = 19.9 months vs. 12.5 months; RR = 0.227, P = 0.001). This suggests that the prognostic impact of MGMT methylation is dependent on therapeutic modalities and schedules. MGMT methylation was not correlated with the main molecular alterations, such as 10q loss and p53 expression.

5-Azacytidine treatment induces demethylation of DAPK1 and MGMT genes and inhibits growth in canine mammary gland tumor cells.

PubMed

Ren, Xiaoli; Li, Huatao; Song, Xianyi; Wu, Yuhong; Liu, Yun

2018-01-01

Canine mammary gland tumors (CMGTs) are the most common, spontaneous types of neoplasias in female dogs. Aberrant DAPK1 and MGMT methylation associated with tumor formation and development in various cancers. 5-Azacytidine is a known specific demethylation drug that covalently binds to DNA methyltransferase. However, the methylation of the DAPK1 and MGMT is unknown with respect to CMGTs. Therefore, we sought to demonstrate the effects of 5-azacytidine on the proliferation of CMGTs cell, and elucidate the potential molecular mechanisms of action in these cancerous cells. The effects of 5-azacytidine on CHMm and CHMp cell proliferation were evaluated by MTT assay. The DAPK1 and MGMT gene methylation patterns in CHMm and CHMp cells and CMGTs blood/tissue samples were analyzed by MSP assay. Effect of 5-azacytidine on the methylation of DAPK1 and MGMT gene, and DAPK1 and MGMT mRNA expression in CHMm and CHMp cells were analyzed by MSP assay and qRT-PCR assay, respectively. 5-Azacytidine may suppress the proliferation of CHMm and CHMp cells. Furthermore, the DAPK1 and MGMT genes were hypermethylated in CHMm/CHMp cells and clinical malignant tumor samples, but not in normal female dogs' blood and tissue. However, the DAPK1 and MGMT genes were re-inducible in CHMm and CHMp cells treated with 5 μM 5-azacytidine. Meanwhile, 5-azacytidine increased the expression of DAPK1 and MGMT mRNA. These results suggest that DAPK1 and MGMT methylation can serve as sensitive diagnostic biomarkers and therapeutic targets for CMGTs. 5-Azacytidine also could be a potential therapeutic candidate for CMGTs.

Elimination of Cancer Stem-Like Cells and Potentiation of Temozolomide Sensitivity by Honokiol in Glioblastoma Multiforme Cells

PubMed Central

Lai, I-Chun; Shih, Ping-Hsiao; Yao, Chih-Jung; Yeh, Chi-Tai; Wang-Peng, Jacqueline; Lui, Tai-Ngar; Chuang, Suang-En; Hu, Tsai-Shu; Lai, Tung-Yuan; Lai, Gi-Ming

2015-01-01

Glioblastoma multiforme (GBM) is the most common adult malignant glioma with poor prognosis due to the resistance to radiotherapy and chemotherapy, which might be critically involved in the repopulation of cancer stem cells (CSCs) after treatment. We had investigated the characteristics of cancer stem-like side population (SP) cells sorted from GBM cells, and studied the effect of Honokiol targeting on CSCs. GBM8401 SP cells possessed the stem cell markers, such as nestin, CD133 and Oct4, and the expressions of self-renewal related stemness genes, such as SMO, Notch3 and IHH (Indian Hedgehog). Honokiol inhibited the proliferation of both GBM8401 parental cells and SP cells in a dose-dependent manner, the IC50 were 5.3±0.72 and 11±1.1 μM, respectively. The proportions of SP in GBM8401 cells were diminished by Honokiol from 1.5±0.22% down to 0.3±0.02% and 0.2±0.01% at doses of 2.5 μM and 5 μM, respectively. The SP cells appeared to have higher expression of O 6-methylguanine-DNA methyltransferase (MGMT) and be more resistant to Temozolomide (TMZ). The resistance to TMZ could be only slightly reversed by MGMT inhibitor O 6-benzylguanine (O 6-BG), but markedly further enhanced by Honokiol addition. Such significant enhancement was accompanied with the higher induction of apoptosis, greater down-regulation of Notch3 as well as its downstream Hes1 expressions in SP cells. Our data indicate that Honokiol might have clinical benefits for the GBM patients who are refractory to TMZ treatment. PMID:25763821

Determination of the methylation status of MGMT in different regions within glioblastoma multiforme.

PubMed

Hamilton, Mark G; Roldán, Gloria; Magliocco, Anthony; McIntyre, John B; Parney, Ian; Easaw, Jacob C

2011-04-01

Epigenetic silencing of the MGMT gene through promoter methylation correlates with improved survival in Glioblastoma Multiforme (GBM) patients receiving concurrent chemoradiotherapy. Although the clinical benefit is primarily seen in patients with methylated MGMT promoter, some unmethylated patients also respond to Temozolomide. One possible explanation may be intratumoral heterogeneity. This study was designed to assess the methylation status of the MGMT promoter in different areas of GBM and determine if methylation status varied depending on the fixation technique (paraffin-embedding versus fresh frozen) used to store tissue. Using intraoperative navigation, biopsies were obtained from three distinct regions: the enhancing outer area, the non-enhancing inner core, and an area immediately outside the enhancing region. Only patients with GBM were included for evaluation and analysis. Samples taken from each area were divided with half stored by flash freezing and the other half stored using paraffin fixation. Methylation Specific-PCR (MS-PCR) was used for analysis of MGMT promoter methylation. Thirteen patients were included. Ten were male with a median age of 62 years. In each patient, samples were taken from the enhancing rim and the necrotic centre. However, it was not considered safe or feasible to obtain samples from the area immediately adjacent to the enhancing tumor rim in one case. All patients were homogeneous for methylation status throughout their tumor and tissue taken adjacent to it when frozen tissue was used. However, four patients had discrepancies in the MGMT promoter status between the frozen and paraffin-embedded blocks and one patient was not homogeneous within the tumor when paraffin-embedded tissue was used. MGMT promoter methylation status was homogeneous in all GBM tumors. Our observation that methylation status varied depending if the DNA was extracted from paraffin-embedded versus frozen tissue is concerning. Although the reason for

Evolution of novel O-methyltransferases from the Vanilla planifolia caffeic acid O-methyltransferase.

PubMed

Li, Huaijun Michael; Rotter, David; Hartman, Thomas G; Pak, Fulya E; Havkin-Frenkel, Daphna; Belanger, Faith C

2006-06-01

The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences.

MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults.

PubMed

Alvarez, Marisa C; Santos, Juliana C; Maniezzo, Nathália; Ladeira, Marcelo S; da Silva, Artur L C; Scaletsky, Isabel C A; Pedrazzoli, José; Ribeiro, Marcelo L

2013-05-28

To evaluate the association between Helicobacter pylori (H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSP-PCR) in gastric biopsy samples from uninfected or H. pylori-infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ(2) test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t-test. Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori-infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori-infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was

Digital PCR quantification of MGMT methylation refines prediction of clinical benefit from alkylating agents in glioblastoma and metastatic colorectal cancer.

PubMed

Barault, L; Amatu, A; Bleeker, F E; Moutinho, C; Falcomatà, C; Fiano, V; Cassingena, A; Siravegna, G; Milione, M; Cassoni, P; De Braud, F; Rudà, R; Soffietti, R; Venesio, T; Bardelli, A; Wesseling, P; de Witt Hamer, P; Pietrantonio, F; Siena, S; Esteller, M; Sartore-Bianchi, A; Di Nicolantonio, F

2015-09-01

O(6)-methyl-guanine-methyl-transferase (MGMT) silencing by promoter methylation may identify cancer patients responding to the alkylating agents dacarbazine or temozolomide. We evaluated the prognostic and predictive value of MGMT methylation testing both in tumor and cell-free circulating DNA (cfDNA) from plasma samples using an ultra-sensitive two-step digital PCR technique (methyl-BEAMing). Results were compared with two established techniques, methylation-specific PCR (MSP) and Bs-pyrosequencing. Thresholds for MGMT methylated status for each technique were established in a training set of 98 glioblastoma (GBM) patients. The prognostic and the predictive value of MGMT methylated status was validated in a second cohort of 66 GBM patients treated with temozolomide in which methyl-BEAMing displayed a better specificity than the other techniques. Cutoff values of MGMT methylation specific for metastatic colorectal cancer (mCRC) tissue samples were established in a cohort of 60 patients treated with dacarbazine. In mCRC, both quantitative assays methyl-BEAMing and Bs-pyrosequencing outperformed MSP, providing better prediction of treatment response and improvement in progression-free survival (PFS) (P < 0.001). Ability of methyl-BEAMing to identify responding patients was validated in a cohort of 23 mCRC patients treated with temozolomide and preselected for MGMT methylated status according to MSP. In mCRC patients treated with dacarbazine, exploratory analysis of cfDNA by methyl-BEAMing showed that MGMT methylation was associated with better response and improved median PFS (P = 0.008). Methyl-BEAMing showed high reproducibility, specificity and sensitivity and was applicable to formalin-fixed paraffin-embedded tissues and cfDNA. This study supports the quantitative assessment of MGMT methylation for clinical purposes since it could refine prediction of response to alkylating agents. © The Author 2015. Published by Oxford University Press on behalf of the European

Focus on Fotemustine.

PubMed

De Rossi, A; Rossi, L; Laudisi, A; Sini, V; Toppo, L; Marchesi, F; Tortorelli, G; Leti, M; Turriziani, M; Aquino, A; Bonmassar, E; De Vecchis, L; Torino, F

2006-12-01

Fotemustine is a cytotoxic alkylating agent, belonging to the group of nitrosourea family. Its mechanism of action is similar to that of other nitrosoureas, characterized by a mono-functional/bi-functional alkylating activity. Worth of consideration is the finding that the presence of high levels of the DNA repair enzyme O6-methylguanine-DNA-methyltransferase (MGMT) in cancer cells confers drug resistance. In different clinical trials Fotemustine showed a remarkable antitumor activity as single agent, and in association with other antineoplastic compounds or treatment modalities. Moreover, its toxicity is generally considered acceptable. The drug has been employed in the treatment of metastatic melanoma, and, on the basis of its pharmacokinetic properties, in brain tumors, either primitive or metastatic. Moreover, Fotemustine shows pharmacodynamic properties similar to those of mono-functional alkylating compounds (e.g. DNA methylating drugs, such as Temozolomide), that have been recently considered for the management of acute refractory leukaemia. Therefore, it is reasonable to assume that this agent could be a good candidate to play a potential role in haematological malignancies.

Structural characterization of the mitomycin 7-O-methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Shanteri; Chang, Aram; Goff, Randal D.

2014-10-02

Mitomycins are quinone-containing antibiotics, widely used as antitumor drugs in chemotherapy. Mitomycin-7-O-methyltransferase (MmcR), a key tailoring enzyme involved in the biosynthesis of mitomycin in Streptomyces lavendulae, catalyzes the 7-O-methylation of both C9{beta}- and C9{alpha}-configured 7-hydroxymitomycins. We have determined the crystal structures of the MmcR-S-adenosylhomocysteine (SAH) binary complex and MmcR-SAH-mitomycin A (MMA) ternary complex at resolutions of 1.9 and 2.3 {angstrom}, respectively. The study revealed MmcR to adopt a common S-adenosyl-L-methionine-dependent O-methyltransferase fold and the presence of a structurally conserved active site general acid-base pair is consistent with a proton-assisted methyltransfer common to most methyltransferases. Given the importance of C7 alkylationmore » to modulate mitomycin redox potential, this study may also present a template toward the future engineering of catalysts to generate uniquely bioactive mitomycins.« less

Leveraging molecular datasets for biomarker-based clinical trial design in glioblastoma.

PubMed

Tanguturi, Shyam K; Trippa, Lorenzo; Ramkissoon, Shakti H; Pelton, Kristine; Knoff, David; Sandak, David; Lindeman, Neal I; Ligon, Azra H; Beroukhim, Rameen; Parmigiani, Giovanni; Wen, Patrick Y; Ligon, Keith L; Alexander, Brian M

2017-07-01

Biomarkers can improve clinical trial efficiency, but designing and interpreting biomarker-driven trials require knowledge of relationships among biomarkers, clinical covariates, and endpoints. We investigated these relationships across genomic subgroups of glioblastoma (GBM) within our institution (DF/BWCC), validated results in The Cancer Genome Atlas (TCGA), and demonstrated potential impacts on clinical trial design and interpretation. We identified genotyped patients at DF/BWCC, and clinical associations across 4 common GBM genomic biomarker groups were compared along with overall survival (OS), progression-free survival (PFS), and survival post-progression (SPP). Significant associations were validated in TCGA. Biomarker-based clinical trials were simulated using various assumptions. Epidermal growth factor receptor (EGFR)(+) and p53(-) subgroups were more likely isocitrate dehydrogenase (IDH) wild-type. Phosphatidylinositol-3 kinase (PI3K)(+) patients were older, and patients with O6-DNA methylguanine-methyltransferase (MGMT)-promoter methylation were more often female. OS, PFS, and SPP were all longer for IDH mutant and MGMT methylated patients, but there was no independent prognostic value for other genomic subgroups. PI3K(+) patients had shorter PFS among IDH wild-type tumors, however, and no DF/BWCC long-term survivors were either EGFR(+) (0% vs 7%, P = .014) or p53(-) (0% vs 10%, P = .005). The degree of biomarker overlap impacted the efficiency of Bayesian-adaptive clinical trials, while PFS and OS distribution variation had less impact. Biomarker frequency was proportionally associated with sample size in all designs. We identified several associations between GBM genomic subgroups and clinical or molecular prognostic covariates and validated known prognostic factors in all survival periods. These results are important for biomarker-based trial design and interpretation of biomarker-only and nonrandomized trials. © The Author(s) 2017. Published by

[Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust].

PubMed

Shen, M L; He, Z N; Zhang, X; Duan, H W; Niu, Y; Bin, P; Ye, M; Meng, T; Dai, Y F; Yu, S F; Chen, W; Zheng, Y X

2017-06-06

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median ( P (25)- P (75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group ( P< 0.001). DNA methylation levels of P16, RASSF1A and MGMT were 2.04±0.41, 2.19 (1.94-2.51), 2.22 (1.94-2.46)%5mC in exposure group, and 2.19±0.40, 2.41 (2.11-2.67), 2.44 (2.15-2.91)%5mC in control group. DNA methylation levels were lower in exposure group ( P values were 0.005, 0.002 and 0.001, respectively). Spearman correlation analysis showed that DNA methylation levels of P16, RASSF1A, and MGMT were negative associated with urinary εdA level ( r values were -0.155, -0.137, and -0.198, respectively, P< 0.05). No significant correlation was observed

Monolignol 4-O-methyltransferases and uses thereof

DOEpatents

Liu, Chang-Jun; Bhuiya, Mohammad-Wadud; Zhang, Kewei

2014-11-18

Modified (iso)eugenol 4-O-methyltransferase enzymes having novel capacity for methylation of monolignols and reduction of lignin polymerization in plant cell wall are disclosed. Sequences encoding the modified enzymes are disclosed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belinsky, Steven A; Palmisano, William A

A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection ofmore » lung and other cancers.« less

Nested methylation-specific polymerase chain reaction cancer detection method

DOEpatents

Belinsky, Steven A [Albuquerque, NM; Palmisano, William A [Edgewood, NM

2007-05-08

A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

In vivo Selection of Autologous MGMT Gene-Modified Cells Following Reduced Intensity Conditioning with BCNU and Temozolomide in the Dog Model

PubMed Central

Gori, Jennifer L.; Beard, Brian C.; Ironside, Christina; Karponi, Garyfalia; Kiem, Hans-Peter

2012-01-01

Chemotherapy with BCNU and temozolomide (TMZ) is commonly used for the treatment of glioblastoma multiforme (GBM) and other cancers. In preparation for a clinical gene therapy study in patients with glioblastoma, we wished to study whether these reagents could be used as a reduced-intensity conditioning regimen for autologous transplantation of gene-modified cells. We used an MGMT(P140K)-expressing lentivirus vector to modify dog CD34+ cells and tested in 4 dogs whether these autologous cells engraft and provide chemoprotection after transplantation. Treatment with O6-benzylguanine (O6BG)/TMZ after transplantation resulted in gene marking levels up to 75%, without significant hematopoietic cytopenia, which is consistent with hematopoietic chemoprotection. Retrovirus integration analysis showed that multiple clones contribute to hematopoiesis. These studies demonstrate the ability to achieve stable engraftment of MGMT(P140K)-modified autologous HSCs after a novel reduced-intensity conditioning protocol using a combination of BCNU and TMZ. Furthermore, we show that MGMT(P140K)-HSC engraftment provides chemoprotection during TMZ dose escalation. Clinically, chemoconditioning with BCNU and TMZ should facilitate engraftment of MGMT(P140K)-modified cells while providing anti-tumor activity for patients with poor prognosis glioblastoma or alkylating agent sensitive tumors, thereby supporting dose-intensified chemotherapy regimens. PMID:22627392

Radiation necrosis presenting as pseudoprogression (PsP) during alectinib treatment of previously radiated brain metastases in ALK-positive NSCLC: Implications for disease assessment and management.

PubMed

Ou, Sai-Hong Ignatius; Klempner, Samuel J; Azada, Michele C; Rausei-Mills, Veronica; Duma, Christopher

2015-06-01

Radiation necrosis presenting as pseudoprogression (PsP) is relatively common after radiation and temozolomide (TMZ) treatment in glioblastoma multiforme (GBM), especially among patients with GBM that harbors intrinsic increased responsiveness to TMZ (methylated O6-methylguanine-DNA methyltransferase [MGMT] promoter). Alectinib is a second generation ALK inhibitor that has significant CNS activity against brain metastases in anaplastic lymphoma kinase (ALK)-rearranged (ALK+) non-small cell lung cancer (NSCLC) patients. We report 2 ALK+ NSCLC patients who met RECIST criteria for progressive disease by central radiologic review due to increased in size from increased contrast enhancement in previously stereotactically radiated brain metastases with ongoing extra-cranial response to alectinib. In both patients alectinib was started within 4 months of completing stereotactic radiosurgery (SRS). The enlarging lesions in both patients were resected and found to have undergone extensive necrosis with no residual tumor pathologically. PsP was incorrectly classified as progressive disease even by central independent imaging review. Treatment-related necrosis of previously SRS-treated brain metastasis during alectinib treatment can present as PsP. It may be impossible to distinguish PsP from true disease progression without a pathologic examination from resected sample. High degree of clinical suspicion, close monitoring and more sensitive imaging modalities may be needed to distinguish PsP versus progression in radiated brain lesions during alectinib treatment especially if there is no progression extra-cranially. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

IGF-1R inhibition induces schedule-dependent sensitization of human melanoma to temozolomide

PubMed Central

Ramcharan, Roger; Aleksic, Tamara; Kamdoum, Wilfride Petnga; Gao, Shan; Pfister, Sophia X.; Tanner, Jordan; Bridges, Esther; Asher, Ruth; Watson, Amanda J.; Margison, Geoffrey P.; Woodcock, Mick; Repapi, Emmanouela; Li, Ji-Liang; Middleton, Mark R.; Macaulay, Valentine M.

2015-01-01

Prior studies implicate type 1 IGF receptor (IGF-1R) in mediating chemo-resistance. Here, we investigated whether IGF-1R influences response to temozolomide (TMZ), which generates DNA adducts that are removed by O6-methylguanine-DNA methyltransferase (MGMT), or persist causing replication-associated double-strand breaks (DSBs). Initial assessment in 10 melanoma cell lines revealed that TMZ resistance correlated with MGMT expression (r = 0.79, p = 0.009), and in MGMT-proficient cell lines, with phospho-IGF-1R (r = 0.81, p = 0.038), suggesting that TMZ resistance associates with IGF-1R activation. Next, effects of IGF-1R inhibitors (IGF-1Ri) AZ3801 and linsitinib (OSI-906) were tested on TMZ-sensitivity, cell cycle progression and DSB induction. IGF-1Ri sensitized BRAF wild-type and mutant melanoma cells to TMZ in vitro, an effect that was independent of MGMT. Cells harboring wild-type p53 were more sensitive to IGF-1Ri, and showed schedule-dependent chemo-sensitization that was most effective when IGF-1Ri followed TMZ. This sequence sensitized to clinically-achievable TMZ concentrations and enhanced TMZ-induced apoptosis. Simultaneous or prior IGF-1Ri caused less effective chemo-sensitization, associated with increased G1 population and reduced accumulation of TMZ-induced DSBs. Clinically relevant sequential (TMZ → IGF-1Ri) treatment was tested in mice bearing A375M (V600E BRAF, wild-type p53) melanoma xenografts, achieving peak plasma/tumor IGF-1Ri levels comparable to clinical Cmax, and inducing extensive intratumoral apoptosis. TMZ or IGF-1Ri caused minor inhibition of tumor growth (gradient reduction 13%, 25% respectively), while combination treatment caused supra-additive growth delay (72%) that was significantly different from control (p < 0.01), TMZ (p < 0.01) and IGF-1Ri (p < 0.05) groups. These data highlight the importance of scheduling when combining IGF-1Ri and other targeted agents with drugs that induce replication-associated DNA damage. PMID

MGMT-STP27 methylation status as predictive marker for response to PCV in anaplastic Oligodendrogliomas and Oligoastrocytomas. A report from EORTC study 26951.

PubMed

van den Bent, Martin J; Erdem-Eraslan, Lale; Idbaih, Ahmed; de Rooi, Johan; Eilers, Paul H C; Spliet, Wim G M; den Dunnen, Wilfred F A; Tijssen, Cees; Wesseling, Pieter; Sillevis Smitt, Peter A E; Kros, Johan M; Gorlia, Thierry; French, Pim J

2013-10-01

The long-term follow-up results from the EORTC-26951 trial showed that the addition of procarbazine, CCNU, and vincristine (PCV) after radiotherapy increases survival in anaplastic oligodendrogliomas/oligoastrocytomas (AOD/AOA). However, some patients appeared to benefit more from PCV treatment than others. We conducted genome-wide methylation profiling of 115 samples included in the EORTC-26951 trial and extracted the CpG island hypermethylated phenotype (CIMP) and MGMT promoter methylation (MGMT-STP27) status. We first show that methylation profiling can be conducted on archival tissues with a performance that is similar to snap-frozen tissue samples. We then conducted methylation profiling on EORTC-26951 clinical trial samples. Univariate analysis indicated that CIMP+ or MGMT-STP27 methylated tumors had an improved survival compared with CIMP- and/or MGMT-STP27 unmethylated tumors [median overall survival (OS), 1.05 vs. 6.46 years and 1.06 vs. 3.8 years, both P < 0.0001 for CIMP and MGMT-STP27 status, respectively]. Multivariable analysis indicates that CIMP and MGMT-STP27 are significant prognostic factors for survival in presence of age, sex, performance score, and review diagnosis in the model. CIMP+ and MGMT-STP27 methylated tumors showed a clear benefit from adjuvant PCV chemotherapy: the median OS of CIMP+ samples in the RT and RT-PCV arms was 3.27 and 9.51 years, respectively (P = 0.0033); for MGMT-STP27 methylated samples, it was 1.98 and 8.65 years. There was no such benefit for CIMP- or for MGMT-STP27 unmethylated tumors. MGMT-STP27 status remained significant in an interaction test (P = 0.003). Statistical analysis of microarray (SAM) identified 259 novel CpGs associated with treatment response. MGMT-STP27 may be used to guide treatment decisions in this tumor type. ©2013 AACR.

Multifuntional Nanotherapeutics for the Combinatorial Drug and Gene Therapy in the Treatment of Glioblastoma Multiforme

NASA Astrophysics Data System (ADS)

Hourigan, Breanne

Glioblastoma multiforme (GBM), a grade IV glioma, is the most common primary brain tumor, affecting about 3 out of 100,000 persons per year in the United States. GBM accounts for about 80% of primary malignant brain tumors, and is also the most aggressive of malignant brain tumors. With exhaustive treatment, survival only averages between 12 and 15 months, with a 2-year survival rate less than 25%. New therapeutic strategies are necessary to improve the outcomes of this disease. Chemotherapy with temozolomide (TMZ), a DNA alkylating agent, is used as a first-line of treatment for GBM. However, GBM tumors develop resistance to TMZ over time due to increased expression of O6-methylguanine-DNA methyltransferase (MGMT), a gene responsible for DNA repair. We previously developed cationic, amphiphilic copolymer poly(lactide-co-glycolide)-g-polyethylenimine (PgP) and demonstrated its utility for nucleic acid delivery. Here, we examine the ability of PgP polyplexes to overcome TMZ resistance and improve therapeutic efficacy through combination drug and gene therapy for GBM treatment. In this study, we evaluated the ability of PgP to deliver siRNA targeting to MGMT (siMGMT), a gene responsible for drug resistance in GBM. Our results demonstrated that PgP effectively forms stable complex with siRNA and protects siRNAs from heparin competition assay, serum- and ribonuclease-mediated degradation, confirming the potential of the polyplex for in vivo delivery. Results from MTT assays showed that PgP/siRNA polyplexes exhibited minimal cytotoxicity compared to untreated cells when incubated with T98G human GBM cells. We also demonstrated that PgP/siMGMT polyplexes mediate knockdown of MGMT protein as well as a significant ˜56% and ˜68% knockdown of MGMT mRNA in T98G GBM cells compared to cells treated with PgP complexed with non-targeting siRNA (siNT) at a 60:1 and 80:1 nitrogen:phosphate (N:P) ratio, respectively. Further, co-incubation of PgP/siMGMT polyplexes with TMZ

Structure of the human gene encoding the protein repair L-isoaspartyl (D-aspartyl) O-methyltransferase.

PubMed

DeVry, C G; Tsai, W; Clarke, S

1996-11-15

The protein L-isoaspartyl/D-aspartyl O-methyltransferase (EC 2.1.1.77) catalyzes the first step in the repair of proteins damaged in the aging process by isomerization or racemization reactions at aspartyl and asparaginyl residues. A single gene has been localized to human chromosome 6 and multiple transcripts arising through alternative splicing have been identified. Restriction enzyme mapping, subcloning, and DNA sequence analysis of three overlapping clones from a human genomic library in bacteriophage P1 indicate that the gene spans approximately 60 kb and is composed of 8 exons interrupted by 7 introns. Analysis of intron/exon splice junctions reveals that all of the donor and acceptor splice sites are in agreement with the mammalian consensus splicing sequence. Determination of transcription initiation sites by primer extension analysis of poly(A)+ mRNA from human brain identifies multiple start sites, with a major site 159 nucleotides upstream from the ATG start codon. Sequence analysis of the 5'-untranslated region demonstrates several potential cis-acting DNA elements including SP1, ETF, AP1, AP2, ARE, XRE, CREB, MED-1, and half-palindromic ERE motifs. The promoter of this methyltransferase gene lacks an identifiable TATA box but is characterized by a CpG island which begins approximately 723 nucleotides upstream of the major transcriptional start site and extends through exon 1 and into the first intron. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for this methyltransferase activity.

Deep-Learning Convolutional Neural Networks Accurately Classify Genetic Mutations in Gliomas.

PubMed

Chang, P; Grinband, J; Weinberg, B D; Bardis, M; Khy, M; Cadena, G; Su, M-Y; Cha, S; Filippi, C G; Bota, D; Baldi, P; Poisson, L M; Jain, R; Chow, D

2018-05-10

The World Health Organization has recently placed new emphasis on the integration of genetic information for gliomas. While tissue sampling remains the criterion standard, noninvasive imaging techniques may provide complimentary insight into clinically relevant genetic mutations. Our aim was to train a convolutional neural network to independently predict underlying molecular genetic mutation status in gliomas with high accuracy and identify the most predictive imaging features for each mutation. MR imaging data and molecular information were retrospectively obtained from The Cancer Imaging Archives for 259 patients with either low- or high-grade gliomas. A convolutional neural network was trained to classify isocitrate dehydrogenase 1 ( IDH1 ) mutation status, 1p/19q codeletion, and O6-methylguanine-DNA methyltransferase ( MGMT ) promotor methylation status. Principal component analysis of the final convolutional neural network layer was used to extract the key imaging features critical for successful classification. Classification had high accuracy: IDH1 mutation status, 94%; 1p/19q codeletion, 92%; and MGMT promotor methylation status, 83%. Each genetic category was also associated with distinctive imaging features such as definition of tumor margins, T1 and FLAIR suppression, extent of edema, extent of necrosis, and textural features. Our results indicate that for The Cancer Imaging Archives dataset, machine-learning approaches allow classification of individual genetic mutations of both low- and high-grade gliomas. We show that relevant MR imaging features acquired from an added dimensionality-reduction technique demonstrate that neural networks are capable of learning key imaging components without prior feature selection or human-directed training. © 2018 by American Journal of Neuroradiology.

Diethylnitrosamine-induced hepatocarcinogenesis is suppressed in lecithin:retinol acyltransferase-deficient mice primarily through retinoid actions immediately after carcinogen administration.

PubMed

Shirakami, Yohei; Gottesman, Max E; Blaner, William S

2012-02-01

Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.

The O-methyltransferase PMT2 mediates methylation of pinosylvin in Scots pine.

PubMed

Paasela, Tanja; Lim, Kean-Jin; Pietiäinen, Milla; Teeri, Teemu H

2017-06-01

Heartwood extractives are important determinants of the natural durability of pine heartwood. The most important phenolic compounds affecting durability are the stilbenes pinosylvin and its monomethylether, which in addition have important functions as phytoalexins in active defense. A substantial portion of the synthesized pinosylvin is 3-methoxylated but the O-methyltransferase responsible for this modification has not been correctly identified. We studied the expression of the stilbene pathway during heartwood development as well as in response to wounding of xylem and UV-C treatment of needles. We isolated and enzymatically characterized a novel O-methyltransferase, PMT2. The methylated product was verified as pinosylvin monomethylether using ultra performance liquid chromatography-tandem mass spectrometry and high performance liquid chromatography analyses. The PMT2 enzyme was highly specific for stilbenes as substrate, in contrast to caffeoyl-CoA O-methyltransferase (CCoAOMT) and PMT1 that were multifunctional. Expression profile and multifunctional activity of CCoAOMT suggest that it might have additional roles outside lignin biosynthesis. PMT1 is not involved in the stilbene pathway and its biological function remains an open question. We isolated a new specific O-methyltransferase responsible for 3-methoxylation of pinosylvin. Expression of PMT2 closely follows stilbene biosynthesis during developmental and stress induction. We propose that PMT2 is responsible for pinosylvin methylation in Scots pine (Pinus sylvestris), instead of the previously characterized methyltransferase, PMT1. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

mRNA Cap Methyltransferase, RNMT-RAM, Promotes RNA Pol II-Dependent Transcription.

PubMed

Varshney, Dhaval; Lombardi, Olivia; Schweikert, Gabriele; Dunn, Sianadh; Suska, Olga; Cowling, Victoria H

2018-05-01

mRNA cap addition occurs early during RNA Pol II-dependent transcription, facilitating pre-mRNA processing and translation. We report that the mammalian mRNA cap methyltransferase, RNMT-RAM, promotes RNA Pol II transcription independent of mRNA capping and translation. In cells, sublethal suppression of RNMT-RAM reduces RNA Pol II occupancy, net mRNA synthesis, and pre-mRNA levels. Conversely, expression of RNMT-RAM increases transcription independent of cap methyltransferase activity. In isolated nuclei, recombinant RNMT-RAM stimulates transcriptional output; this requires the RAM RNA binding domain. RNMT-RAM interacts with nascent transcripts along their entire length and with transcription-associated factors including the RNA Pol II subunits SPT4, SPT6, and PAFc. Suppression of RNMT-RAM inhibits transcriptional markers including histone H2BK120 ubiquitination, H3K4 and H3K36 methylation, RNA Pol II CTD S5 and S2 phosphorylation, and PAFc recruitment. These findings suggest that multiple interactions among RNMT-RAM, RNA Pol II factors, and RNA along the transcription unit stimulate transcription. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Arabidopsis Serrate Coordinates Histone Methyltransferases ATXR5/6 and RNA Processing Factor RDR6 to Regulate Transposon Expression.

PubMed

Ma, Zeyang; Castillo-González, Claudia; Wang, Zhiye; Sun, Di; Hu, Xiaomei; Shen, Xuefeng; Potok, Magdalena E; Zhang, Xiuren

2018-06-18

Serrate (SE) is a key component in RNA metabolism. Little is known about whether and how it can regulate epigenetic silencing. Here, we report histone methyltransferases ATXR5 and ATXR6 (ATXR5/6) as novel partners of SE. ATXR5/6 deposit histone 3 lysine 27 monomethylation (H3K27me1) to promote heterochromatin formation, repress transposable elements (TEs), and control genome stability in Arabidopsis. SE binds to ATXR5/6-regulated TE loci and promotes H3K27me1 accumulation in these regions. Furthermore, SE directly enhances ATXR5 enzymatic activity in vitro. Unexpectedly, se mutation suppresses the TE reactivation and DNA re-replication phenotypes in the atxr5 atxr6 mutant. The suppression of TE expression results from triggering RNA-dependent RNA polymerase 6 (RDR6)-dependent RNA silencing in the se atxr5 atxr6 mutant. We propose that SE facilitates ATXR5/6-mediated deposition of the H3K27me1 mark while inhibiting RDR6-mediated RNA silencing to protect TE transcripts. Hence, SE coordinates epigenetic silencing and RNA processing machineries to fine-tune the TE expression. Copyright © 2018 Elsevier Inc. All rights reserved.

DGKI methylation status modulates the prognostic value of MGMT in glioblastoma patients treated with combined radio-chemotherapy with temozolomide.

PubMed

Etcheverry, Amandine; Aubry, Marc; Idbaih, Ahmed; Vauleon, Elodie; Marie, Yannick; Menei, Philippe; Boniface, Rachel; Figarella-Branger, Dominique; Karayan-Tapon, Lucie; Quillien, Veronique; Sanson, Marc; de Tayrac, Marie; Delattre, Jean-Yves; Mosser, Jean

2014-01-01

Consistently reported prognostic factors for glioblastoma (GBM) are age, extent of surgery, performance status, IDH1 mutational status, and MGMT promoter methylation status. We aimed to integrate biological and clinical prognostic factors into a nomogram intended to predict the survival time of an individual GBM patient treated with a standard regimen. In a previous study we showed that the methylation status of the DGKI promoter identified patients with MGMT-methylated tumors that responded poorly to the standard regimen. We further evaluated the potential prognostic value of DGKI methylation status. 399 patients with newly diagnosed GBM and treated with a standard regimen were retrospectively included in this study. Survival modelling was performed on two patient populations: intention-to-treat population of all included patients (population 1) and MGMT-methylated patients (population 2). Cox proportional hazard models were fitted to identify the main prognostic factors. A nomogram was developed for population 1. The prognostic value of DGKI promoter methylation status was evaluated on population 1 and population 2. The nomogram-based stratification of the cohort identified two risk groups (high/low) with significantly different median survival. We validated the prognostic value of DGKI methylation status for MGMT-methylated patients. We also demonstrated that the DGKI methylation status identified 22% of poorly responding patients in the low-risk group defined by the nomogram. Our results improve the conventional MGMT stratification of GBM patients receiving standard treatment. These results could help the interpretation of published or ongoing clinical trial outcomes and refine patient recruitment in the future.

Promoter hypermethylation of CDKN2A, MGMT, MLH1, and DAPK genes in laryngeal squamous cell carcinoma and their associations with clinical profiles of the patients.

PubMed

Pierini, Stefano; Jordanov, Stanislav H; Mitkova, Atanaska V; Chalakov, Ivan J; Melnicharov, Mincho B; Kunev, Kuncho V; Mitev, Vanio I; Kaneva, Radka P; Goranova, Teodora E

2014-08-01

Laryngeal squamous cell carcinoma (laryngeal SCC) is a frequently occurring cancer of the head and neck area. Epigenetic changes of tumor-related genes contribute to its genesis and progression. We assessed promoter methylation status of the selected genes (CDKN2A, MGMT, MLH1, and DAPK) using methylation-sensitive high resolution melting (MS-HRM) in 100 patients with laryngeal SCC and studied the correlations with clinical characteristics. The prevalence of promoter methylation in MGMT, CDKN2A, MLH1, and DAPK was 59 of 97 (60.8%), 46 of 97 (47.4%), 45 of 97 (46.4%), and 41 of 97 patients (42.3%), respectively. Significantly increased methylation of CDKN2A was observed in heavy smokers. Epigenetic inactivation of CDKN2A and MLH1 were found to be associated with lymph node involvement. An inverse correlation was present between MLH1 methylation and alcohol consumption. Our results strongly suggest that deregulation of p16-associated, and MLH1-associated pathways, because of promoter hypermethylation, is associated with increased cancer cell migration, tumor invasiveness, and, thus, aggressive phenotype. Copyright © 2013 Wiley Periodicals, Inc.

2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase

PubMed Central

Dong, Hongping; Chang, David C.; Hua, Maggie Ho Chia; Lim, Siew Pheng; Chionh, Yok Hian; Hia, Fabian; Lee, Yie Hou; Kukkaro, Petra; Lok, Shee-Mei; Dedon, Peter C.; Shi, Pei-Yong

2012-01-01

RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m7GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of

Purification and Characterization of S-Adenosyl-l-Methionine: Desoxyhemigossypol-6-O-Methyltransferase from Cotton Plants. An Enzyme Capable of Methylating the Defense Terpenoids of Cotton1

PubMed Central

Liu, Jinggao; Benedict, Chauncey R.; Stipanovic, Robert D.; Bell, Alois A.

1999-01-01

Cotton contains a unique group of terpenoids including desoxyhemigossypol, hemigossypol, gossypol, hemigossypolone, and the heliocides that are part of the plant's defense system against pathogenic fungi and insects. Desoxyhemigossypol is a key intermediate in the biosynthesis of these compounds. We have isolated, purified, and characterized from cotton stele tissue infected with Verticillium dahliae a methyltransferase (S-adenosyl-l-Met: desoxyhemigossypol-6-O-methyltransferase) that specifically methylates the 6-position of desoxyhemigossypol to form desoxyhemigossypol-6-methyl ether with a Km value of 4.5 μm for desoxyhemigossypol and a Kcat/Km of 5.08 × 104 s−1 (mol/L)−1. The molecular mass of the native enzyme is 81.4 kD and is dissociated into two subunits of 41.2 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The enzymatic reaction does not require Mg+2 and is inhibited 98% with 10 mm p-chloromercuribenzoate. Desoxyhemigossypol-6-methyl ether leads to the biosynthesis of methylated hemigossypol, gossypol, hemigossypolone, and the heliocides, which lowers their effectiveness as phytoalexins and insecticides. PMID:10557251

DNA Damage Induced by Alkylating Agents and Repair Pathways

PubMed Central

Kondo, Natsuko; Takahashi, Akihisa; Ono, Koji; Ohnishi, Takeo

2010-01-01

The cytotoxic effects of alkylating agents are strongly attenuated by cellular DNA repair processes, necessitating a clear understanding of the repair mechanisms. Simple methylating agents form adducts at N- and O-atoms. N-methylations are removed by base excision repair, AlkB homologues, or nucleotide excision repair (NER). O6-methylguanine (MeG), which can eventually become cytotoxic and mutagenic, is repaired by O6-methylguanine-DNA methyltransferase, and O6MeG:T mispairs are recognized by the mismatch repair system (MMR). MMR cannot repair the O6MeG/T mispairs, which eventually lead to double-strand breaks. Bifunctional alkylating agents form interstrand cross-links (ICLs) which are more complex and highly cytotoxic. ICLs are repaired by complex of NER factors (e.g., endnuclease xeroderma pigmentosum complementation group F-excision repair cross-complementing rodent repair deficiency complementation group 1), Fanconi anemia repair, and homologous recombination. A detailed understanding of how cells cope with DNA damage caused by alkylating agents is therefore potentially useful in clinical medicine. PMID:21113301

Permeability Surface Area Product Using Perfusion Computed Tomography Is a Valuable Prognostic Factor in Glioblastomas Treated with Radiotherapy Plus Concomitant and Adjuvant Temozolomide.

PubMed

Saito, Taiichi; Sugiyama, Kazuhiko; Ikawa, Fusao; Yamasaki, Fumiyuki; Ishifuro, Minoru; Takayasu, Takeshi; Nosaka, Ryo; Nishibuchi, Ikuno; Muragaki, Yoshihiro; Kawamata, Takakazu; Kurisu, Kaoru

2017-01-01

The current standard treatment protocol for patients with newly diagnosed glioblastoma (GBM) includes surgery, radiotherapy, and concomitant and adjuvant temozolomide (TMZ). We hypothesized that the permeability surface area product (PS) from a perfusion computed tomography (PCT) study is associated with sensitivity to TMZ. The aim of this study was to determine whether PS values were correlated with prognosis of GBM patients who received the standard treatment protocol. This study included 36 patients with GBM that were newly diagnosed between October 2005 and September 2014 and who underwent preoperative PCT study and the standard treatment protocol. We measured the maximum value of relative cerebral blood volume (rCBVmax) and the maximum PS value (PSmax). We statistically examined the relationship between PSmax and prognosis using survival analysis, including other clinicopathologic factors (age, Karnofsky performance status [KPS], extent of resection, O6-methylguanine-DNA methyltransferase [MGMT] status, second-line use of bevacizumab, and rCBVmax). Log-rank tests revealed that age, KPS, MGMT status, and PSmax were significantly correlated with overall survival. Multivariate analysis using the Cox regression model showed that PSmax was the most significant prognostic factor. Receiver operating characteristic curve analysis showed that PSmax had the highest accuracy in differentiating longtime survivors (LTSs) (surviving more than 2 years) from non-LTSs. At a cutoff point of 8.26 mL/100 g/min, sensitivity and specificity were 90% and 70%, respectively. PSmax from PCT study can help predict survival time in patients with GBM receiving the standard treatment protocol. Survival may be related to sensitivity to TMZ. Copyright © 2016 Elsevier Inc. All rights reserved.

The association between MTHFR 677C>T genotype and folate status and genomic and gene-specific DNA methylation in the colon of individuals without colorectal neoplasia.

PubMed

Hanks, Joanna; Ayed, Iyeman; Kukreja, Neil; Rogers, Chris; Harris, Jessica; Gheorghiu, Alina; Liu, Chee Ling; Emery, Peter; Pufulete, Maria

2013-12-01

Decreased genomic and increased gene-specific DNA methylation predispose to colorectal cancer. Dietary folate intake and the methylenetetrahydrofolate reductase polymorphism (MTHFR 677C>T) may influence risk by modifying DNA methylation. We investigated the associations between MTHFR 677C>T genotype, folate status, and DNA methylation in the colon. We conducted a cross-sectional study of 336 men and women (age 19-92 y) in the United Kingdom without colorectal neoplasia. We obtained blood samples for measurement of serum and red blood cell folate, plasma homocysteine, and MTHFR 677C>T genotype and colonic tissue biopsies for measurement of colonic tissue folate and DNA methylation (genomic- and gene-specific, estrogen receptor 1, ESR1; myoblast determination protein 1, MYOD1; insulin-like growth factor II, IGF2; tumor suppressor candidate 33, N33; adenomatous polyposis coli, APC; mut-L homolog 1, MLH1; and O(6)-methylguanine-DNA methyltransferase, MGMT) by liquid chromatography/electrospray ionization mass spectrometry and pyrosequencing, respectively. Of the 336 subjects recruited, 185 (55%) carried the CC, 119 (35%) the CT, and 32 (10%) the TT alleles. No significant differences in systemic markers of folate status and colonic tissue folate between genotypes were found. The MTHFR TT genotype was not associated with genomic or gene-specific DNA methylation. Biomarkers of folate status were not associated with genomic DNA methylation. Relations between biomarkers of folate status and gene-specific methylation were inconsistent. However, low serum folate was associated with high MGMT methylation (P = 0.001). MTHFR 677C>T genotype and folate status were generally not associated with DNA methylation in the colon of a folate-replete population without neoplasia.

The Value of 5-Aminolevulinic Acid in Low-grade Gliomas and High-grade Gliomas Lacking Glioblastoma Imaging Features: An Analysis Based on Fluorescence, Magnetic Resonance Imaging, 18F-Fluoroethyl Tyrosine Positron Emission Tomography, and Tumor Molecular Factors.

PubMed

Jaber, Mohammed; Wölfer, Johannes; Ewelt, Christian; Holling, Markus; Hasselblatt, Martin; Niederstadt, Thomas; Zoubi, Tarek; Weckesser, Matthias; Stummer, Walter

2016-03-01

Approximately 20% of grade II and most grade III gliomas fluoresce after 5-aminolevulinic acid (5-ALA) application. Conversely, approximately 30% of nonenhancing gliomas are actually high grade. The aim of this study was to identify preoperative factors (ie, age, enhancement, 18F-fluoroethyl tyrosine positron emission tomography [F-FET PET] uptake ratios) for predicting fluorescence in gliomas without typical glioblastomas imaging features and to determine whether fluorescence will allow prediction of tumor grade or molecular characteristics. Patients harboring gliomas without typical glioblastoma imaging features were given 5-ALA. Fluorescence was recorded intraoperatively, and biopsy specimens collected from fluorescing tissue. World Health Organization (WHO) grade, Ki-67/MIB-1 index, IDH1 (R132H) mutation status, O-methylguanine DNA methyltransferase (MGMT) promoter methylation status, and 1p/19q co-deletion status were assessed. Predictive factors for fluorescence were derived from preoperative magnetic resonance imaging and F-FET PET. Classification and regression tree analysis and receiver-operating-characteristic curves were generated for defining predictors. Of 166 tumors, 82 were diagnosed as WHO grade II, 76 as grade III, and 8 as glioblastomas grade IV. Contrast enhancement, tumor volume, and F-FET PET uptake ratio >1.85 predicted fluorescence. Fluorescence correlated with WHO grade (P < .001) and Ki-67/MIB-1 index (P < .001), but not with MGMT promoter methylation status, IDH1 mutation status, or 1p19q co-deletion status. The Ki-67/MIB-1 index in fluorescing grade III gliomas was higher than in nonfluorescing tumors, whereas in fluorescing and nonfluorescing grade II tumors, no differences were noted. Age, tumor volume, and F-FET PET uptake are factors predicting 5-ALA-induced fluorescence in gliomas without typical glioblastoma imaging features. Fluorescence was associated with an increased Ki-67/MIB-1 index and high-grade pathology. Whether

Characterization of a candidate tetravalent vaccine based on 2'-O-methyltransferase mutants

PubMed Central

Züst, Roland; Li, Shi-Hua; Xie, Xuping; Velumani, Sumathy; Chng, Melissa; Toh, Ying-Xiu; Zou, Jing; Dong, Hongping; Shan, Chao; Pang, Jassia; Qin, Cheng-Feng; Newell, Evan W.; Shi, Pei-Yong

2018-01-01

Dengue virus (DENV) is one of the most widespread arboviruses. The four DENV serotypes infect about 400 million people every year, causing 96 million clinical dengue cases, of which approximately 500’000 are severe and potentially life-threatening. The only licensed vaccine has a limited efficacy and is only recommended in regions with high endemicity. We previously reported that 2’-O-methyltransferase mutations in DENV-1 and DENV-2 block their capacity to inhibit type I IFNs and render the viruses attenuated in vivo, making them amenable as vaccine strains; here we apply this strategy to all four DENV serotypes to generate a tetravalent, non-chimeric live-attenuated dengue vaccine. 2’-O-methyltransferase mutants of all four serotypes are highly sensitive to type I IFN inhibition in human cells. The tetravalent formulation is attenuated and immunogenic in mice and cynomolgus macaques and elicits a response that protects from virus challenge. These results show the potential of 2’-O-methyltransferase mutant viruses as a safe, tetravalent, non-chimeric dengue vaccine. PMID:29298302

Biosynthesis of estragole and methyl-eugenol in sweet basil (Ocimum basilicum L). Developmental and chemotypic association of allylphenol O-methyltransferase activities.

PubMed

Lewinsohn, E; Ziv-Raz, I; Dudai, N; Tadmor, Y; Lastochkin, E; Larkov, O; Chaimovitsh, D; Ravid, U; Putievsky, E; Pichersky, E; Shoham, Y

2000-12-07

Sweet basil (Ocimum basilicum L., Lamiaceae) is a common herb, used for culinary and medicinal purposes. The essential oils of different sweet basil chemotypes contain various proportions of the allyl phenol derivatives estragole (methyl chavicol), eugenol, and methyl eugenol, as well as the monoterpene alcohol linalool. To monitor the developmental regulation of estragole biosynthesis in sweet basil, an enzymatic assay for S-adenosyl-L-methionine (SAM):chavicol O-methyltransferase activity was developed. Young leaves display high levels of chavicol O-methyltransferase activity, but the activity was negligible in older leaves, indicating that the O-methylation of chavicol primarily occurs early during leaf development. The O-methyltransferase activities detected in different sweet basil genotypes differed in their substrate specificities towards the methyl acceptor substrate. In the high-estragole-containing chemotype R3, the O-methyltransferase activity was highly specific for chavicol, while eugenol was virtually not O-methylated. In contrast, chemotype 147/97, that contains equal levels of estragole and methyl eugenol, displayed O-methyltransferase activities that accepted both chavicol and eugenol as substrates, generating estragole and methyl eugenol, respectively. Chemotype SW that contains high levels of eugenol, but lacks both estragole and methyl eugenol, had apparently no allylphenol dependent O-methyltransferase activities. These results indicate the presence of at least two types of allylphenol-specific O-methyltransferase activities in sweet basil chemotypes, one highly specific for chavicol; and a different one that can accept eugenol as a substrate. The relative availability and substrate specificities of these O-methyltransferase activities biochemically rationalizes the variation in the composition of the essential oils of these chemotypes.

A simple quantitative diagnostic alternative for MGMT DNA-methylation testing on RCL2 fixed paraffin embedded tumors using restriction coupled qPCR.

PubMed

Pulverer, Walter; Hofner, Manuela; Preusser, Matthias; Dirnberger, Elisabeth; Hainfellner, Johannes A; Weinhaeusel, Andreas

2014-01-01

MGMT promoter methylation is associated with favorable prognosis and chemosensitivity in glioblastoma multiforme (GBM), especially in elderly patients. We aimed to develop a simple methylation-sensitive restriction enzyme (MSRE)-based quantitative PCR (qPCR) assay, allowing the quantification of MGMT promoter methylation. DNA was extracted from non-neoplastic brain (n = 24) and GBM samples (n = 20) upon 3 different sample conservation conditions (-80 °C, formalin-fixed and paraffin-embedded (FFPE); RCL2-fixed). We evaluated the suitability of each fixation method with respect to the MSRE-coupled qPCR methylation analyses. Methylation data were validated by MALDITOF. qPCR was used for evaluation of alternative tissue conservation procedures. DNA from FFPE tissue failed reliable testing; DNA from both RCL2-fixed and fresh frozen tissues performed equally well and was further used for validation of the quantitative MGMT methylation assay (limit of detection (LOD): 19.58 pg), using individual's undigested sample DNA for calibration. MGMT methylation analysis in non-neoplastic brain identified a background methylation of 0.10 ± 11% which we used for defining a cut-off of 0.32% for patient stratification. Of GBM patients 9 were MGMT methylationpositive (range: 0.56 - 91.95%), and 11 tested negative. MALDI-TOF measurements resulted in a concordant classification of 94% of GBM samples in comparison to qPCR. The presented methodology allows quantitative MGMT promoter methylation analyses. An amount of 200 ng DNA is sufficient for triplicate analyses including control reactions and individual calibration curves, thus excluding any DNA qualityderived bias. The combination of RCL2-fixation and quantitative methylation analyses improves pathological routine examination when histological and molecular analyses on limited amounts of tumor samples are necessary for patient stratification.

Mechanisms of chemoresistance to alkylating agents in malignant glioma.

PubMed

Sarkaria, Jann N; Kitange, Gaspar J; James, C David; Plummer, Ruth; Calvert, Hilary; Weller, Michael; Wick, Wolfgang

2008-05-15

Intrinsic or acquired chemoresistance to alkylating agents is a major cause of treatment failure in patients with malignant brain tumors. Alkylating agents, the mainstay of treatment for brain tumors, damage the DNA and induce apoptosis, but the cytotoxic activity of these agents is dependent on DNA repair pathways. For example, O6-methylguanine DNA adducts can cause double-strand breaks, but this is dependent on a functional mismatch repair pathway. Thus, tumor cell lines deficient in mismatch repair are resistant to alkylating agents. Perhaps the most important mechanism of resistance to alkylating agents is the DNA repair enzyme O6-methylguanine methyltransferase, which can eliminate the cytotoxic O6-methylguanine DNA adduct before it causes harm. Another mechanism of resistance to alkylating agents is the base excision repair (BER) pathway. Consequently, efforts are ongoing to develop effective inhibitors of BER. Poly(ADP-ribose)polymerase plays a pivotal role in BER and is an important therapeutic target. Developing effective strategies to overcome chemoresistance requires the identification of reliable preclinical models that recapitulate human disease and which can be used to facilitate drug development. This article describes the diverse mechanisms of chemoresistance operating in malignant glioma and efforts to develop reliable preclinical models and novel pharmacologic approaches to overcome resistance to alkylating agents.

The search for a melanoma-tailored chemotherapy in the new era of personalized therapy: a phase II study of chemo-modulating temozolomide followed by fotemustine and a cooperative study of GOIM (Gruppo Oncologico Italia Meridionale).

PubMed

Guida, Michele; Tommasi, Stefania; Strippoli, Sabino; Natalicchio, Maria Iole; De Summa, Simona; Pinto, Rosamaria; Cramarossa, Antonio; Albano, Anna; Pisconti, Salvatore; Aieta, Michele; Ridolfi, Ruggiero; Azzariti, Amalia; Guida, Gabriella; Lorusso, Vito; Colucci, Giusepe

2018-05-10

It is frequently asked whether chemotherapy can still play a role in metastatic melanoma considering the effectiveness of the available drugs today, including antiCTLA4/antiPD1 immunotherapy and antiBRAF/antiMEK inhibitors. However, only approximately half of patients respond to these drugs, and the majority progress after 6-11 months. Therefore, a need for other therapeutic options is still very much apparent. We report the first large trial of a sequential full dose of fotemustine (FM) preceded by a low dose of temozolomide (TMZ) as a chemo-modulator in order to inactivate the DNA repair action of O(6)-methylguanine DNA-methyltransferase (MGMT). Primary endpoints were overall response and safety. We also evaluated specific biological parameters aiming to tailor these chemotherapies to selected patients. A total of 69 consecutive patients were enrolled. The main features included a median age of 60 years (21-81) and M1c stage, observed in 74% of the patients, with brain metastases in 15% and high LDH levels in 42% of the patients. The following schedule was used: oral TMZ 100 mg/m 2 on days 1 and 2 and FM iv 100 mg/m 2 on day 2, 4 h after TMZ; A translational study aiming to analyse MGMT methylation status and base-excision repair (BER) gene expression was performed in a subset of 14 patients. We reported an overall response rate of 30.3% with 3 complete responses and a disease control rate of 50.5%. The related toxicity rate was low and mainly of haematological types. Although our population had a very poor prognosis, we observed a PFS of 6 months and an OS of 10 months. A non-significant correlation with response was found with the mean expression level of the three genes involved in the BER pathway (APE1, XRCC1 and PARP1), whereas no association was found with MGMT methylation status. This schedule could represent a good alternative for patients who are not eligible for immune or targeted therapy or whose previous therapies have failed. EUDRACT 2009

Methyltransferase That Modifies Guanine 966 of the 16 S rRNA: FUNCTIONAL IDENTIFICATION AND TERTIARY STRUCTURE*

PubMed Central

Lesnyak, Dmitry V.; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V.; Bogdanov, Alexey A.; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A.

2010-01-01

N2-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m2G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m2G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m2G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05 Å. The structure closely resembles RsmC rRNA methyltransferase, specific for m2G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m2G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed. PMID:17189261

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

PubMed

Lesnyak, Dmitry V; Osipiuk, Jerzy; Skarina, Tatiana; Sergiev, Petr V; Bogdanov, Alexey A; Edwards, Aled; Savchenko, Alexei; Joachimiak, Andrzej; Dontsova, Olga A

2007-02-23

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.

Dihydromethysticin (DHM) Blocks Tobacco Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Induced O6-Methylguanine in a Manner Independent of the Aryl Hydrocarbon Receptor (AhR) Pathway in C57BL/6 Female Mice.

PubMed

Narayanapillai, Sreekanth C; Lin, Shang-Hsuan; Leitzman, Pablo; Upadhyaya, Pramod; Baglole, Carolyn J; Xing, Chengguo

2016-11-21

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a key carcinogen responsible for tobacco smoke-induced lung carcinogenesis. Among the types of DNA damage caused by NNK and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), O 6 -methylguanine (O 6 -mG) is likely the most carcinogen in A/J mice. Results of our previous studies showed that levels of O 6 -mG and other types of NNAL-derived DNA damage were preferentially reduced in the lung of female A/J mice upon dietary treatment with dihydromethysticin (DHM), a promising lung cancer chemopreventive agent from kava. Such a differential blockage may be mediated via an increased level of NNAL glucuronidation, thereby leading to its detoxification. The potential of the aryl hydrocarbon receptor (AhR) as an upstream target of DHM mediating these events was evaluated herein using Ahr +/- and Ahr -/- C57BL/6 female mice because DHM was reported as an AhR agonist. DHM (0.05, 0.2, and 1.0 mg/g of diet) and dihydrokavain (DHK, an inactive analogue, 1.0 mg/g of diet) were given to mice for 7 days, followed by a single intraperitoneal dose of NNK at 100 mg/kg of body weight. The effects of DHM on the amount of O 6 -mG in the lung, on the urinary ratio of glucuronidated NNAL (NNAL-Gluc) and free NNAL, and on CYP1A1/2 activity in the liver microsomes were analyzed. As observed in A/J mice, DHM treatment significantly and dose-dependently reduced the level of O 6 -mG in the target lung tissue, but there were no significant differences in O 6 -mG reduction between mice from Ahr +/- and Ahr -/- backgrounds. Similarly, in both strains, DHM at 1 mg/g of diet significantly increased the urinary ratio of NNAL-Gluc to free NNAL and CYP1A1/2 enzymatic activity in liver with no changes detected at lower DHM dosages. Because none of these effects of DHM were dependent on Ahr status, AhR clearly is not the upstream target for DHM.

Plant isoflavone and isoflavanone O-methyltransferase genes

DOEpatents

Broeckling, Bettina E.; Liu, Chang-Jun; Dixon, Richard A.

2014-08-19

The invention provides enzymes that encode O-methyltransferases (OMTs) from Medicago truncatula that allow modification to plant (iso)flavonoid biosynthetic pathways. In certain aspects of the invention, the genes encoding these enzymes are provided. The invention therefore allows the modification of plants for isoflavonoid content. Transgenic plants comprising such enzymes are also provided, as well as methods for improving disease resistance in plants. Methods for producing food and nutraceuticals, and the resulting compositions, are also provided.

Crystal structure of norcoclaurine-6-O-methyltransferase, a key rate-limiting step in the synthesis of benzylisoquinoline alkaloids.

PubMed

Robin, Adeline Y; Giustini, Cécile; Graindorge, Matthieu; Matringe, Michel; Dumas, Renaud

2016-09-01

Growing pharmaceutical interest in benzylisoquinoline alkaloids (BIA) coupled with their chemical complexity make metabolic engineering of microbes to create alternative platforms of production an increasingly attractive proposition. However, precise knowledge of rate-limiting enzymes and negative feedback inhibition by end-products of BIA metabolism is of paramount importance for this emerging field of synthetic biology. In this work we report the structural characterization of (S)-norcoclaurine-6-O-methyltransferase (6OMT), a key rate-limiting step enzyme involved in the synthesis of reticuline, the final intermediate to be shared between the different end-products of BIA metabolism, such as morphine, papaverine, berberine and sanguinarine. Four different crystal structures of the enzyme from Thalictrum flavum (Tf 6OMT) were solved: the apoenzyme, the complex with S-adenosyl-l-homocysteine (SAH), the complexe with SAH and the substrate and the complex with SAH and a feedback inhibitor, sanguinarine. The Tf 6OMT structural study provides a molecular understanding of its substrate specificity, active site structure and reaction mechanism. This study also clarifies the inhibition of Tf 6OMT by previously suggested feedback inhibitors. It reveals its high and time-dependent sensitivity toward sanguinarine. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

Endogenous 5-methylcytosine protects neighboring guanines from N7 and O6-methylation and O6-pyridyloxobutylation by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

PubMed

Ziegel, Rebecca; Shallop, Anthony; Upadhyaya, Pramod; Jones, Roger; Tretyakova, Natalia

2004-01-20

All CG dinucleotides along exons 5-8 of the p53 tumor suppressor gene contain endogenous 5-methylcytosine (MeC). These same sites (e.g., codons 157, 158, 245, 248, and 273) are mutational hot spots in smoking-induced lung cancer. Several groups used the UvrABC endonuclease incision assay to demonstrate that methylated CG dinucleotides of the p53 gene are the preferred binding sites for the diol epoxides of bay region polycyclic aromatic hydrocarbons (PAH). In contrast, effects of endogenous cytosine methylation on the distribution of DNA lesions induced by tobacco-specific nitrosamines, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have not been elucidated. In the work presented here, a stable isotope labeling HPLC-ESI-MS/MS approach was employed to analyze the reactivity of the N7 and O6 positions of guanines within hemimethylated and fully methylated CG dinucleotides toward NNK-derived methylating and pyridyloxobutylating species. 15N3-labeled guanine bases were placed within synthetic DNA sequences representing endogenously methylated p53 codons 154, 157, and 248, followed by treatment with acetylated precursors to NNK diazohydroxides. HPLC-ESI-MS/MS analysis was used to determine the relative yields of N7- and O6-guanine adducts at the 15N3-labeled position. In all cases, the presence of MeC inhibited the formation of N7-methylguanine, O6-methylguanine, and O6-pyridyloxobutylguanine at a neighboring G, with the greatest decrease observed in fully methylated dinucleotides and at guanines preceded by MeC. Furthermore, the O6-Me-dG/N7-Me-G molar ratios were decreased in the presence of the 5'-neighboring MeC, suggesting that the observed decline in O6-alkylguanine adduct yields is, at least partially, a result of an altered reactivity pattern in methylated CG dinucleotides. These results indicate that, unlike N2-guanine adducts of PAH diol epoxides, NNK-induced N7- and O6-alkylguanine adducts are not preferentially formed at the endogenously

In vitro evaluation of combined temozolomide and radiotherapy using X  rays and high-linear energy transfer radiation for glioblastoma.

PubMed

Barazzuol, Lara; Jena, Raj; Burnet, Neil G; Jeynes, Jonathan C G; Merchant, Michael J; Kirkby, Karen J; Kirkby, Norman F

2012-05-01

High-linear energy transfer radiation offers superior biophysical properties over conventional radiotherapy and may have a great potential for treating radioresistant tumors, such as glioblastoma. However, very little pre-clinical data exists on the effects of high-LET radiation on glioblastoma cell lines and on the concomitant application of chemotherapy. This study investigates the in vitro effects of temozolomide in combination with low-energy protons and α particles. Cell survival, DNA damage and repair, and cell growth were examined in four human glioblastoma cell lines (LN18, T98G, U87 and U373) after treatment with either X rays, protons (LET 12.91 keV/μm), or α particles (LET 99.26 keV/μm) with or without concurrent temozolomide at clinically-relevant doses of 25 and 50 μM. The relative biological effectiveness at 10% survival (RBE(10)) increased as LET increased: 1.17 and 1.06 for protons, and 1.84 and 1.68 for α particles in the LN18 and U87 cell lines, respectively. Temozolomide administration increased cell killing in the O(6)-methylguanine DNA methyltransferase-methylated U87 and U373 cell lines. In contrast, temozolomide provided no therapeutic enhancement in the methylguanine DNA methyltransferase-unmethylated LN18 and T98G cell lines. In addition, the residual number of γ-H2AX foci at 24 h after treatment with radiation and concomitant temozolomide was found to be lower than or equal to that expected by DNA damage with either of the individual treatments. Kinetics of foci disappearance after X-ray and proton irradiation followed similar time courses; whereas, loss of γ-H2AX foci after α particle irradiation occurred at a slower rate than that by low-LET radiation (half-life 12.51-16.87 h). The combination of temozolomide with different radiation types causes additive rather than synergistic cytotoxicity. Nevertheless, particle therapy combined with chemotherapy may offer a promising alternative with the additional benefit of superior

Limited role for extended maintenance temozolomide for newly diagnosed glioblastoma.

PubMed

Gramatzki, Dorothee; Kickingereder, Philipp; Hentschel, Bettina; Felsberg, Jörg; Herrlinger, Ulrich; Schackert, Gabriele; Tonn, Jörg-Christian; Westphal, Manfred; Sabel, Michael; Schlegel, Uwe; Wick, Wolfgang; Pietsch, Torsten; Reifenberger, Guido; Loeffler, Markus; Bendszus, Martin; Weller, Michael

2017-04-11

To explore an association with survival of modifying the current standard of care for patients with newly diagnosed glioblastoma of surgery followed by radiotherapy plus concurrent and 6 cycles of maintenance temozolomide chemotherapy (TMZ/RT → TMZ) by extending TMZ beyond 6 cycles. The German Glioma Network cohort was screened for patients with newly diagnosed glioblastoma who received TMZ/RT → TMZ and completed ≥6 cycles of maintenance chemotherapy without progression. Associations of clinical patient characteristics, molecular markers, and residual tumor determined by magnetic resonance imaging after 6 cycles of TMZ with progression-free survival (PFS) and overall survival (OS) were analyzed with the log-rank test. Multivariate analyses using the Cox proportional hazards model were performed to assess associations of prolonged TMZ use with outcome. Sixty-one of 142 identified patients received at least 7 maintenance TMZ cycles (median 11, range 7-20). Patients with extended maintenance TMZ treatment had better PFS (20.5 months, 95% confidence interval [CI] 17.7-23.3, vs 17.2 months, 95% CI 10.2-24.2, p = 0.035) but not OS (32.6 months, 95% CI 28.9-36.4, vs 33.2 months, 95% CI 25.3-41.0, p = 0.126). However, there was no significant association of prolonged TMZ chemotherapy with PFS (hazard ratio [HR] = 0.8, 95% CI 0.4-1.6, p = 0.559) or OS (HR = 1.6, 95% CI 0.8-3.3, p = 0.218) adjusted for age, extent of resection, Karnofsky performance score, presence of residual tumor, O 6 -methylguanine DNA methyltransferase (MGMT) promoter methylation status, or isocitrate dehydrogenase ( IDH ) mutation status. These data may not support the practice of prolonging maintenance TMZ chemotherapy beyond 6 cycles. This study provides Class III evidence that in patients with newly diagnosed glioblastoma, prolonged TMZ chemotherapy does not significantly increase PFS or OS. © 2017 American Academy of Neurology.

A Continuous, Quantitative Fluorescent Assay for Plant Caffeic acid O-Methyltransferases

USDA-ARS?s Scientific Manuscript database

Plant caffeic acid O-methyltransferases (COMTs) use s-adenosylmethionine (ado-met), as a methyl donor to transmethylate their preferred (phenolic) substrates in-vivo, and will generally utilize a range of phenolic compounds in-vitro. Collazo et al. (2005; Analytical Biochemistry 342: 86-92) have pu...

Discovery and characterization of new O-methyltransferase from the genome of the lignin-degrading fungus Phanerochaete chrysosporium for enhanced lignin degradation.

PubMed

Thanh Mai Pham, Le; Kim, Yong Hwan

2016-01-01

Using bioinformatic homology search tools, this study utilized sequence phylogeny, gene organization and conserved motifs to identify members of the family of O-methyltransferases from lignin-degrading fungus Phanerochaete chrysosporium. The heterologous expression and characterization of O-methyltransferases from P. chrysosporium were studied. The expressed protein utilized S-(5'-adenosyl)-L-methionine p-toluenesulfonate salt (SAM) and methylated various free-hydroxyl phenolic compounds at both meta and para site. In the same motif, O-methyltransferases were also identified in other white-rot fungi including Bjerkandera adusta, Ceriporiopsis (Gelatoporia) subvermispora B, and Trametes versicolor. As free-hydroxyl phenolic compounds have been known as inhibitors for lignin peroxidase, the presence of O-methyltransferases in white-rot fungi suggested their biological functions in accelerating lignin degradation in white-rot basidiomycetes by converting those inhibitory groups into non-toxic methylated phenolic ones. Copyright © 2015 Elsevier Inc. All rights reserved.

A Set of Regioselective O-Methyltransferases Gives Rise to the Complex Pattern of Methoxylated Flavones in Sweet Basil1[C][W][OA

PubMed Central

Berim, Anna; Hyatt, David C.; Gang, David R.

2012-01-01

Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/4′-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 4′- and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins’ stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6- and 4′-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation. PMID:22923679

[Cytotoxicity of lysomustine and its isomers, and their potential use for selection of cells].

PubMed

Rozov, F N; Grinenko, T S; Levit, G L; Grishakov, A N; Beliavskiĭ, A V; Krasnov, V P

2011-01-01

N epsilon-Nitroso-N epsilon- [N'-(2-chloroethyl)carbamoyl]-L-lysine (I) and N epsilon- [N'-(2-chloroethyl)-N'-nitrosocarbamoyl]-L-lysine (II), the isomers being the constituents of antitumor agent Lysomustine, were obtained by RFHPLC. The study of cytotoxicity of the above compounds against K562 cells showed that the lesions induced by isomer (II) produce a significant cytotoxic effect but can be efficiently repaired by the action of MGMT (O6-methylaguanine DNA methyltransferase). Under similar conditions, the lesions induced by isomer (I) produce substantially smaller effect but are weakly if at all repairable by MGMT. The effects of a clinically approved agent Lysomustine, which is the mixture of isomers (I) and (II), are similar to those of isomer (II). The results obtained point to a different chemical nature of DNA lesions induced by two Lysomustine isomers. Our data indicate that Lysomustine and its isomer (II) can be used for in vitro selection of cells expressing MGMT.

Hypermethylation of testis derived transcript gene promoter significantly correlates with worse outcomes in glioblastoma patients.

PubMed

Wang, Li-jia; Bai, Yu; Bao, Zhao-shi; Chen, Yan; Yan, Zhuo-hong; Zhang, Wei; Zhang, Quan-geng

2013-01-01

Glioblastoma is the most common and lethal cancer of the central nervous system. Global genomic hypomethylation and some CpG island hypermethylation are common hallmarks of these malignancies, but the effects of these methylation abnormalities on glioblastomas are still largely unclear. Methylation of the O6-methylguanine-DNA methyltransferase promoter is currently an only confirmed molecular predictor of better outcome in temozolomide treatment. To better understand the relationship between CpG island methylation status and patient outcome, this study launched DNA methylation profiles for thirty-three primary glioblastomas (pGBMs) and nine secondary glioblastomas (sGBMs) with the expectation to identify valuable prognostic and therapeutic targets. We evaluated the methylation status of testis derived transcript (TES) gene promoter by microarray analysis of glioblastomas and the prognostic value for TES methylation in the clinical outcome of pGBM patients. Significance analysis of microarrays was used for genes significantly differently methylated between 33 pGBM and nine sGBM. Survival curves were calculated according to the Kaplan-Meier method, and differences between curves were assessed using the log-rank test. Then, we treated glioblastoma cell lines (U87 and U251) with 5-aza-2-deoxycytidines (5-aza-dC) and detected cell biological behaviors. Microarray data analysis identified TES promoter was hypermethylated in pGBMs compared with sGBMs (P < 0.05). Survival curves from the Kaplan-Meier method analysis revealed that the patients with TES hypermethylation had a short overall survival (P < 0.05). This abnormality is also confirmed in glioblastoma cell lines (U87 and U251). Treating these cells with 5-aza-dC released TES protein expression resulted in significant inhibition of cell growth (P = 0.013). Hypermethylation of TES gene promoter highly correlated with worse outcome in pGBM patients. TES might represent a valuable prognostic marker for glioblastoma.

GE-29EXPRESSION SUBCLASS PROFILE IN PSEUDOPROGRESSION AND TRUE PROGRESSION IN NEWLY DIAGNOSED GBM

PubMed Central

Robin, Adam; Raghunathan, Aditya; Leung, Denise; Burmeister, Charlotte; Poisson, Laila; Scarpace, Lisa; Walbert, Tobias; Mikkelsen, Tom; Lee, Ian

2014-01-01

INTRODUCTION: The hallmark of glioblastoma multiforme (GBM) is its penchant for relentless progression. Pseudoprogression describes a post-treatment reaction demonstrating increased edema and contrast enhancement similar to typical tumor progression except that on subsequent imaging without escalation of antitumor therapy these changes stabilize or revert [1]. Accurate identification of pseudoprogression has important implications for therapy and research and potentially prognosis as well. Increased cellular proliferation (Ki-67 indices) and the presence of a methylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter have been associated with higher rates of pseudoprogression [2,3]. However, more sensitive and specific biomarkers of pseudoprogression are needed. This study seeks to identify novel indicators of pseudoprogression. METHODS: Patients were identified using the Hermelin Brain Tumor Center database at Henry Ford Hospital. Tissues from 52 patients with newly diagnosed GBM between 1992 and 2011 were gathered and whole genome sequencing and subtyping was performed by The Cancer Genome Atlas researchers. Retrospective chart review was carried out. Patients were assigned to either pseudoprogression (PP) or true progression (TP) groups based on whether changes suggestive of disease progression on MRI within 2 months of post-operative therapy initiation regressed without additional antitumor therapy during the ensuing 4 months. The incidence of pseudoprogression and GBM subclass were correlated using Fisher's Exact Test. RESULTS: Forty-one of 52 (79%) cases were identified as TP while 11/52 (21%) were found to have PP. In our study population, PP was associated with significantly increased median survival compared with TP (735 versus 313 days, respectively, p < 0.0012). The molecular subclass profile for both groups included a predominance of Mesenchymal and Neural subtypes, revealing no correlation between GBM subclass and the risk of pseudoprogression

Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells.

PubMed

Gou, Qing; He, ShuJiao; Zhou, ZeJian

2017-02-01

Hepatocellular carcinoma is the most common subtype of liver cancer. Protein arginine N-methyltransferase 1 was shown to be upregulated in various cancers. However, the role of protein arginine N-methyltransferase 1 in hepatocellular carcinoma progression remains incompletely understood. We investigated the clinical and functional significance of protein arginine N-methyltransferase 1 in a series of clinical hepatocellular carcinoma samples and a panel of hepatocellular carcinoma cell lines. We performed suppression analysis of protein arginine N-methyltransferase 1 using small interfering RNA to determine the biological roles of protein arginine N-methyltransferase 1 in hepatocellular carcinoma. In addition, the expression of epithelial-mesenchymal transition indicators was verified by western blotting in hepatocellular carcinoma cell lines after small interfering RNA treatment. Protein arginine N-methyltransferase 1 expression was found to be significantly upregulated in hepatocellular carcinoma cell lines and clinical tissues. Moreover, downregulation of protein arginine N-methyltransferase 1 in hepatocellular carcinoma cells by small interfering RNA could inhibit cell proliferation, migration, and invasion in vitro. These results indicate that protein arginine N-methyltransferase 1 may contribute to hepatocellular carcinoma progression and serves as a promising target for the treatment of hepatocellular carcinoma patients.

Biotechnological Production of Dimethoxyflavonoids Using a Fusion Flavonoid O-Methyltransferase Possessing Both 3'- and 7-O-Methyltransferase Activities.

PubMed

Lee, Danbi; Park, Hye Lin; Lee, Sang-Won; Bhoo, Seong Hee; Cho, Man-Ho

2017-05-26

Although they are less abundant in nature, methoxyflavonoids have distinct physicochemical and pharmacological properties compared to common nonmethylated flavonoids. Thus, enzymatic conversion and biotransformation using genetically engineered microorganisms of flavonoids have been attempted for the efficient production of methoxyflavonoids. Because of their regiospecificity, more than two flavonoid O-methyltransferases (FOMTs) and enzyme reactions are required to biosynthesize di(or poly)-methoxyflavonoids. For the one-step biotechnological production of bioactive di-O-methylflavonoids, we generated a multifunctional FOMT fusing a 3'-OMT (SlOMT3) and a 7-OMT (OsNOMT). The SlOMT3/OsNOMT fusion enzyme possessed both 3'- and 7-OMT activities to diverse flavonoid substrates, which were comparable to those of individual SlOMT3 and OsNOMT. The SlOMT3/OsNOMT enzyme also showed 3'- and 7-OMT activity for 7- or 3'-O-methylflavonoids, respectively, suggesting that the fusion enzyme can sequentially methylate flavonoids into di-O-methylflavonoids. The biotransformation of the flavonoids quercetin, luteolin, eriodictyol, and taxifolin using SlOMT3/OsNOMT-transformed Escherichia coli generated corresponding di-O-methylflavonoids, rhamnazin, velutin, 3',7-di-O-methyleriodictyol, and 3',7-di-O-methyltaxifolin, respectively. These results indicate that dimethoxyflavonoids may be efficiently produced from nonmethylated flavonoid precursors through a one-step biotransformation using the engineered E. coli harboring the SlOMT3/OsNOMT fusion gene.

The histone methyltransferase Smyd2 is a negative regulator of macrophage activation by suppressing interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) production.

PubMed

Xu, Guiliang; Liu, Guilin; Xiong, Sidong; Liu, Haiyan; Chen, Xi; Zheng, Biao

2015-02-27

SET and MYND domain-containing 2 (Smyd2), a histone 3 lysine 4- and histone 3 lysine 36 (H3K36)-specific methyltransferase, plays critical roles in cardiac development and tumorigenesis. However, the role of Smyd2 in immunity and inflammation remains poorly understood. In this study, we report that Smyd2 is a novel negative regulator for macrophage activation and M1 polarization. Elevated Smyd2 expression suppresses the production of proinflammatory cytokines, including IL-6 and TNF, and inhibits the expression of important cell surface molecules, including major MHC-II and costimulatory molecules. Furthermore, macrophages with high Smyd2 expression inhibit Th-17 cell differentiation but promote regulatory T cell differentiation as a result of increased TGF-β production and decreased IL-6 secretion. In macrophages, Smyd2 specifically facilitates H3K36 dimethylation at Tnf and Il6 promoters to suppress their transcription and inhibits NF-κB and ERK signaling. Therefore, our data demonstrate that epigenetic modification by Smyd2-mediated H3K36 dimethylation at Tnf and Il6 promoters plays an important role in the regulation of macrophage activation during inflammation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

DNA-methylation analysis identifies the E-cadherin gene as a potential marker of disease progression in patients with monoclonal gammopathies.

PubMed

Seidl, Sonja; Ackermann, Jutta; Kaufmann, Hannes; Keck, Andrea; Nösslinger, Thomas; Zielinski, Christoph C; Drach, Johannes; Zöchbauer-Müller, Sabine

2004-06-15

Silencing of tumor suppressor genes (TSG) by aberrant methylation (referred to as methylation) contributes to the pathogenesis of various human malignancies. However, little is known about the methylation of known and putative TSGs in monoclonal gammopathies. Thus, the authors investigated the methylation frequencies of 10 genes in patients with monoclonal gammopathies. The methylation patterns of the genes p16(INK4a) (p16), tissue inhibitor of metalloproteinase 3 (TIMP3), p15(INK4b) (p15), E-cadherin (ECAD), death-associated protein kinase (DAPK), p73, RAS-association domain family 1A (RASSF1A), p14, O(6)-methylguanine DNA methyltransferase (MGMT), and retinoid acid receptor beta2 (RARbeta) were determined in patients with monoclonal gammopathy of undetermined significance (MGUS; n = 29), smoldering multiple myeloma (SMM; n = 5), multiple myeloma (MM; n = 113), or plasma cell leukemia (PCL; n = 7) by methylation-specific polymerase chain reaction analysis. Methylation frequencies for p16, TIMP3, p15, ECAD, DAPK, p73, RASSF1A, p14, MGMT, and RARbeta were as follows: 28%, 35%, 10%, 0%, 17%, 21%, 14%, 14%, 7%, and 0%, respectively, in patients with MGUS and 36%, 29%, 27%, 27%, 22%, 15%, 15%, 9%, 4%, and 0%, respectively, in patients with MM. Methylation of at least 1 of these genes was detected in 79% of patients with MGUS and in 80% of patients with MM. Although methylation of ECAD was not detected in patients with MGUS, it was observed frequently in patients with MM and with even greater frequency in patients with PCL. It is noteworthy that an association was found between ECAD methylation and poor prognostic markers in patients with MM. Methylation of certain genes can be detected frequently in patients with monoclonal gammopathies. The current data suggest that methylation of ECAD is a marker of disease progression in patients with MM and PCL. Copyright 2004 American Cancer Society.

Clinicopathological observations of colorectal serrated lesions associated with invasive carcinoma and high-grade intraepithelial neoplasm

PubMed Central

XU, SHENG; WANG, LUPING; YANG, GUANGZHI; LI, LIN; WANG, JIN; XU, CHUNWEI; GE, CHANG

2013-01-01

The aim of this study was to investigate the clinicopathological characteristics of colorectal serrated lesions associated with invasive carcinoma and high-grade intraepithelial neoplasm (HIN), as well as to determine the immunohistochemical expression of MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), K-ras and O6-methylguanine-DNA methyltransferase (MGMT). A total of 5,347 cases diagnosed with colorectal polyp or adenoma were included in this study from October 2002 to September 2009. A total of 16 cases of colorectal serrated lesions associated with invasive carcinoma/HIN were screened. These comprised seven cases of traditional serrated adenoma (TSA) associated with invasive carcinoma and HIN, six cases of sessile serrated adenoma (SSA) associated with invasive carcinoma/HIN and three cases of hyperplastic polyp (HP) associated with invasive carcinoma/HIN. TSA associated with invasive carcinoma/HIN predominantly occurred in the rectum with a clearly serrated structure and ectopic crypts. High-grade dysplasia was observed in filiform TSA, which was more prone to carcinogenesis. SSA associated with invasive carcinoma/HIN mainly occurred in the ileocecal junction, with the SSA serrated glands closely located adjacent to the muscularis mucosa and the basal crypt expanded with inverted T- or L-shaped branches. HPs were observed in three cases in the cancer-adjacent tissues with invasive carcinoma, while a HP-SSA/TSA-carcinoma sequence was found in two cases. Immunohistochemistry showed that MGMT expression was significantly different in the serrated lesion tissues compared with that in cancer tissues (P=0.022), control cancer tissues (P=0.002) and normal colorectal epithelial tissues (P=0.003). TSA and SSA may progress to cancer or directly develop into invasive adenocarcinoma. Filiform TSA easily develops into HIN, followed by infiltration. HP may arise from the cancer-adjacent tissues of the invasive carcinoma, which are closely adjacent to the cancer tissues

miR-125b controls apoptosis and temozolomide resistance by targeting TNFAIP3 and NKIRAS2 in glioblastomas

PubMed Central

Haemmig, S; Baumgartner, U; Glück, A; Zbinden, S; Tschan, M P; Kappeler, A; Mariani, L; Vajtai, I; Vassella, E

2014-01-01

Diffusely infiltrating gliomas are among the most prognostically discouraging neoplasia in human. Temozolomide (TMZ) in combination with radiotherapy is currently used for the treatment of glioblastoma (GBM) patients, but less than half of the patients respond to therapy and chemoresistance develops rapidly. Epigenetic silencing of the O6-methylguanine-DNA methyltransferase (MGMT) has been associated with longer survival in GBM patients treated with TMZ, but nuclear factor κB (NF-κB)-mediated survival signaling and TP53 mutations contribute significantly to TMZ resistance. Enhanced NF-κB is in part owing to downregulation of negative regulators of NF-κB activity, including Tumor necrosis factor alpha-induced protein 3 (TNFAIP3) and NF-κB inhibitor interacting RAS-like 2 (NKIRAS2). Here we provide a novel mechanism independent of TP53 and MGMT by which oncogenic miR-125b confers TMZ resistance by targeting TNFAIP3 and NKIRAS2. GBM cells overexpressing miR-125b showed increased NF-κB activity and upregulation of anti-apoptotic and cell cycle genes. This was significantly associated with resistance of GBM cells to TNFα- and TNF-related inducing ligand-induced apoptosis as well as resistance to TMZ. Conversely, overexpression of anti-miR-125b resulted in cell cycle arrest, increased apoptosis and increased sensitivity to TMZ, indicating that endogenous miR-125b is sufficient to control these processes. GBM cells overexpressing TNFAIP3 and NKIRAS2 were refractory to miR-125b-induced apoptosis resistance as well as TMZ resistance, indicating that both genes are relevant targets of miR-125b. In GBM tissues, high miR-125b expression was significantly correlated with nuclear NF-κB confirming that miR-125b is implicated in NF-κB signaling. Most remarkably, miR-125b overexpression was clearly associated with shorter overall survival of patients treated with TMZ, suggesting that this microRNA is an important predictor of response to therapy. PMID:24901050

Molecular cloning and functional expression of a stress-induced multifunctional O-methyltransferase with pinosylvin methyltransferase activity from Scots pine (Pinus sylvestris L.).

PubMed

Chiron, H; Drouet, A; Claudot, A C; Eckerskorn, C; Trost, M; Heller, W; Ernst, D; Sandermann, H

2000-12-01

Formation of pinosylvin (PS) and pinosylvin 3-O-monomethyl ether (PSM), as well as the activities of stilbene synthase (STS) and S-adenosyl-1-methionine (SAM):pinosylvin O-methyltransferase (PMT), were induced strongly in needles of Scots pine seedlings upon ozone treatment, as well as in cell suspension cultures of Scots pine upon fungal elicitation. A SAM-dependent PMT protein was purified and partially characterised. A cDNA encoding PMT was isolated from an ozone-induced Scots pine cDNA library. Southern blot analysis of the genomic DNA suggested the presence of a gene family. The deduced protein sequence showed the typical highly conserved regions of O-methyltransferases (OMTs), and average identities of 20-56% to known OMTs. PMT expressed in Escherichia coli corresponded to that of purified PMT (40 kDa) from pine cell cultures. The recombinant enzyme catalysed the methylation of PS, caffeic acid, caffeoyl-CoA and quercetin. Several other substances, such as astringenin, resveratrol, 5-OH-ferulic acid, catechol and luteolin, were also methylated. Recombinant PMT thus had a relatively broad substrate specificity. Treatment of 7-year old Scots pine trees with ozone markedly increased the PMT mRNA level. Our results show that PMT represents a new SAM-dependent OMT for the methylation of stress-induced pinosylvin in Scots pine needles.

Quercetin-Induced Lifespan Extension in Podospora anserina Requires Methylation of the Flavonoid by the O-Methyltransferase PaMTH1.

PubMed

Warnsmann, Verena; Hainbuch, Saskia; Osiewacz, Heinz D

2018-01-01

Quercetin is a flavonoid that is ubiquitously found in vegetables and fruits. Like other flavonoids, it is active in balancing cellular reactive oxygen species (ROS) levels and has a cyto-protective function. Previously, a link between ROS balancing, aging, and the activity of O -methyltransferases was reported in different organisms including the aging model Podospora anserina. Here we describe a role of the S -adenosylmethionine-dependent O -methyltransferase PaMTH1 in quercetin-induced lifespan extension. We found that effects of quercetin treatment depend on the methylation state of the flavonoid. Specifically, we observed that quercetin treatment increases the lifespan of the wild type but not of the PaMth1 deletion mutant. The lifespan increasing effect is not associated with effects of quercetin on mitochondrial respiration or ROS levels but linked to the induction of the PaMth1 gene. Overall, our data demonstrate a novel role of O -methyltransferase in quercetin-induced longevity and identify the underlying pathway as part of a network of longevity assurance pathways with the perspective to intervene into mechanisms of biological aging.

pH-Dependent Singlet O2 Oxidation Kinetics of Guanine and 9-Methylguanine: An Online Mass Spectrometry and Spectroscopy Study Combined with Theoretical Exploration.

PubMed

Lu, Wenchao; Sun, Yan; Zhou, Wenjing; Liu, Jianbo

2018-01-11

We report a kinetic and mechanistic study on the title reactions, in which 1 O 2 was generated by the reaction of H 2 O 2 with Cl 2 and bubbled into an aqueous solution of guanine and 9-methylguanine (9MG) at different pH values. Oxidation kinetics and product branching ratios were measured using online electrospray ionization mass spectrometry coupled with absorption and emission spectrophotometry, and product structures were determined by collision-induced dissociation (CID) tandem mass spectrometry. Experiments revealed strong pH dependence of the reactions. The oxidation of guanine is noticeable only in basic solution, while the oxidation of 9MG is weak in acidic solution, increases in neutral solution, and becomes intensive in basic solution. 5-Guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) were detected as the major oxidation products of guanine and 9MG, and Sp became dominant in basic solution. A reaction intermediate was captured in mass spectra, and assigned to gem-diol on the basis of CID measurements. This intermediate served as the precursor for the formation of Gh. After taking into account solution compositions at each pH, first-order oxidation rate constants were extracted for individual species: that is, 3.2-3.6 × 10 7 M -1 s -1 for deprotonated guanine, and 1.2 × 10 6 and 4.6-4.9 × 10 7 M -1 s -1 for neutral and deprotonated 9MG, respectively. Guided by approximately spin-projected density-functional-theory-calculated reaction potential energy surfaces, the kinetics for the initial 1 O 2 addition to guanine and 9MG was evaluated using transition state theory (TST). The comparison between TST modeling and experiment confirms that 1 O 2 addition is rate-limiting for oxidation, which forms endoperoxide and peroxide intermediates as determined in previous measurements of the same systems in the gas phase.

Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, C.; Deavours, B; Richard, S

2006-01-01

In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 4'-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for themore » 4'-O-methylation of the isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.« less

Structural Basis for Dual Functionality of Isoflavonoid O-Methyltransferases in the Evolution of Plant Defense Responses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, C.-J.; Deavours, B.E.; Richard, S.B.

2007-07-10

In leguminous plants such as pea (Pisum sativum), alfalfa (Medicago sativa), barrel medic (Medicago truncatula), and chickpea (Cicer arietinum), 4'-O-methylation of isoflavonoid natural products occurs early in the biosynthesis of defense chemicals known as phytoalexins. However, among these four species, only pea catalyzes 3-O-methylation that converts the pterocarpanoid isoflavonoid 6a-hydroxymaackiain to pisatin. In pea, pisatin is important for chemical resistance to the pathogenic fungus Nectria hematococca. While barrel medic does not biosynthesize 6a-hydroxymaackiain, when cell suspension cultures are fed 6a-hydroxymaackiain, they accumulate pisatin. In vitro, hydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) from barrel medic exhibits nearly identical steady state kinetic parameters for themore » 4'-O-methylation of the isoflavonoid intermediate 2,7,4'-trihydroxyisoflavanone and for the 3-O-methylation of the 6a-hydroxymaackiain isoflavonoid-derived pterocarpanoid intermediate found in pea. Protein x-ray crystal structures of HI4'OMT substrate complexes revealed identically bound conformations for the 2S,3R-stereoisomer of 2,7,4'-trihydroxyisoflavanone and the 6aR,11aR-stereoisomer of 6a-hydroxymaackiain. These results suggest how similar conformations intrinsic to seemingly distinct chemical substrates allowed leguminous plants to use homologous enzymes for two different biosynthetic reactions. The three-dimensional similarity of natural small molecules represents one explanation for how plants may rapidly recruit enzymes for new biosynthetic reactions in response to changing physiological and ecological pressures.« less

The histone demethylase KDM5A is a key factor for the resistance to temozolomide in glioblastoma

PubMed Central

Banelli, Barbara; Carra, Elisa; Barbieri, Federica; Würth, Roberto; Parodi, Federica; Pattarozzi, Alessandra; Carosio, Roberta; Forlani, Alessandra; Allemanni, Giorgio; Marubbi, Daniela; Florio, Tullio; Daga, Antonio; Romani, Massimo

2015-01-01

Notwithstanding current multimodal treatment, including surgery, radiotherapy and chemotherapy with temozolomide (TMZ), median survival of glioblastoma (GBM) patients is about 14 months, due to the rapid emergence of cell clones resistant to treatment. Therefore, understanding the mechanisms underlying chemoresistance is mandatory to improve treatments' outcome. We generated TMZ resistant cells (TMZ-R) from a GBM cell line and from cancer stem cell-enriched cultures isolated from human GBMs. We demonstrated that TMZ resistance is partially reverted by “drug wash-out” suggesting the contribution of epigenetic mechanisms in drug resistance and supporting the possibility of TMZ rechallenge in GBM patients after prior drug exposure. The expression of histone lysine demethylase genes (KDMs) was increased in TMZ-R cells compared to parental cells, and TMZ resistance or restored sensitivity was mimicked by over-expressing or inactivating KDM5A. Methylation and expression of O6-methylguanine-DNA methyltransferase (MGMT) and drug efflux mechanisms were not altered in TMZ-R cells compared to parental TMZ sensitive cells. TMZ-R cells transiently acquired morphologic and molecular characteristics of differentiated tumor cells, features that were lost after drug wash-out. In conclusion, we demonstrated that treatment-induced TMZ resistance in GBM involves epigenetic mechanisms in a subset of slow-cycling and transiently partially differentiated cells that escape drug cytotoxicity, overcome G2 checkpoint and sustain clonal growth. We found that TMZ-R cells are sensitive to histone deacethylase inhibitors (HDACi) that synergize with TMZ. This strong synergism could be exploited to develop novel combined adjuvant therapies for this rapidly progressing and invariably lethal cancer. PMID:26566863

The histone demethylase KDM5A is a key factor for the resistance to temozolomide in glioblastoma.

PubMed

Banelli, Barbara; Carra, Elisa; Barbieri, Federica; Würth, Roberto; Parodi, Federica; Pattarozzi, Alessandra; Carosio, Roberta; Forlani, Alessandra; Allemanni, Giorgio; Marubbi, Daniela; Florio, Tullio; Daga, Antonio; Romani, Massimo

2015-01-01

Notwithstanding current multimodal treatment, including surgery, radiotherapy and chemotherapy with temozolomide (TMZ), median survival of glioblastoma (GBM) patients is about 14 months, due to the rapid emergence of cell clones resistant to treatment. Therefore, understanding the mechanisms underlying chemoresistance is mandatory to improve treatments' outcome. We generated TMZ resistant cells (TMZ-R) from a GBM cell line and from cancer stem cell-enriched cultures isolated from human GBMs. We demonstrated that TMZ resistance is partially reverted by "drug wash-out" suggesting the contribution of epigenetic mechanisms in drug resistance and supporting the possibility of TMZ rechallenge in GBM patients after prior drug exposure. The expression of histone lysine demethylase genes (KDMs) was increased in TMZ-R cells compared to parental cells, and TMZ resistance or restored sensitivity was mimicked by over-expressing or inactivating KDM5A. Methylation and expression of O6-methylguanine-DNA methyltransferase (MGMT) and drug efflux mechanisms were not altered in TMZ-R cells compared to parental TMZ sensitive cells. TMZ-R cells transiently acquired morphologic and molecular characteristics of differentiated tumor cells, features that were lost after drug wash-out. In conclusion, we demonstrated that treatment-induced TMZ resistance in GBM involves epigenetic mechanisms in a subset of slow-cycling and transiently partially differentiated cells that escape drug cytotoxicity, overcome G2 checkpoint and sustain clonal growth. We found that TMZ-R cells are sensitive to histone deacethylase inhibitors (HDACi) that synergize with TMZ. This strong synergism could be exploited to develop novel combined adjuvant therapies for this rapidly progressing and invariably lethal cancer.

Association of Catechol-O-Methyltransferase (COMT) Polymorphism and Academic Achievement in a Chinese Cohort

ERIC Educational Resources Information Center

Yeh, Ting-Kuang; Chang, Chun-Yen; Hu, Chung-Yi; Yeh, Ting-Chi; Lin, Ming-Yeh

2009-01-01

Catechol-O-methyltransferase (COMT) is a methylation enzyme that catalyzes the degradation pathway and inactivation of dopamine. It is accepted widely as being involved in the modulation of dopaminergic physiology and prefrontal cortex (PFC) function. The COMT Val158Met polymorphism is associated with variation in COMT activity. COMT 158Met allele…

Glutamine synthetase expression as a valuable marker of epilepsy and longer survival in newly diagnosed glioblastoma multiforme

PubMed Central

Rosati, Anna; Poliani, Pietro Luigi; Todeschini, Alice; Cominelli, Manuela; Medicina, Daniela; Cenzato, Marco; Simoncini, Edda Lucia; Magrini, Stefano Maria; Buglione, Michela; Grisanti, Salvatore; Padovani, Alessandro

2013-01-01

Abstract Background Glutamine synthetase (GS) is an astrocytic enzyme catalyzing the conversion of glutamate and ammonia to glutamine. Its up-regulation has been related to higher tumor proliferation and poor prognosis in extra-cerebral tumors. We have previously reported a GS deficiency in patients with glioblastoma multiforme (GBM) who also developed epilepsy, which is a favorable prognostic factor in glioma. Here, we investigated the prognostic value of GS expression in patients with GBM with or without epilepsy and its correlation with survival. Methods We conducted a clinical and histopathological study on 83 (52 males) consecutive patients with newly diagnosed GBM. Immunohistochemical expression of GS was scored semi-quantitatively on the basis of cell number, staining intensity, and distribution of immunoreactive cells. Several clinical and neuropathological variables were analyzed in relation to survival and GS expression. Results Median age at diagnosis was 62 years. At the last evaluation, with a median follow-up of 11.5 months (range, 1.5–58 months), 5 patients (6%) were still alive and 78 (94%) were dead. GS expression patterns in neoplastic cells were inversely correlated to the presence of epilepsy (P < .0001 for intensity and P < .009 for homogeneity of GS distribution, respectively). Univariate analysis showed that RPA score, epilepsy, O6-methylguanine-DNA methyltransferase (MGM)T status, application of Stupp protocol, and GS intensity pattern had a significant impact on survival. Absent/low intensity of GS expression was significantly associated with a longer survival in both uni- (19 vs 8 months; P < .0005) and multivariate (P = .003) analyses. Conclusions Absent/low-intensity GS expression pattern represents a valuable biomarker of both epilepsy and overall survival in GBM. PMID:23410662

Structure and function of flavivirus NS5 methyltransferase.

PubMed

Zhou, Yangsheng; Ray, Debashish; Zhao, Yiwei; Dong, Hongping; Ren, Suping; Li, Zhong; Guo, Yi; Bernard, Kristen A; Shi, Pei-Yong; Li, Hongmin

2007-04-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m7GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA-->m7GpppA-->m7GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m7GpppA-RNA can be readily methylated to m7GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 A resolution showed a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K61-D146-K182-E218 motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.

Structure and Function of Flavivirus NS5 Methyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou,Y.; Ray, D.; Zhao, Y.

2007-01-01

The plus-strand RNA genome of flavivirus contains a 5' terminal cap 1 structure (m{sup 7}GpppAmG). The flaviviruses encode one methyltransferase, located at the N-terminal portion of the NS5 protein, to catalyze both guanine N-7 and ribose 2'-OH methylations during viral cap formation. Representative flavivirus methyltransferases from dengue, yellow fever, and West Nile virus (WNV) sequentially generate GpppA {yields} m{sup 7}GpppA {yields} m{sup 7}GpppAm. The 2'-O methylation can be uncoupled from the N-7 methylation, since m{sup 7}GpppA-RNA can be readily methylated to m{sup 7}GpppAm-RNA. Despite exhibiting two distinct methylation activities, the crystal structure of WNV methyltransferase at 2.8 {angstrom} resolution showedmore » a single binding site for S-adenosyl-L-methionine (SAM), the methyl donor. Therefore, substrate GpppA-RNA should be repositioned to accept the N-7 and 2'-O methyl groups from SAM during the sequential reactions. Electrostatic analysis of the WNV methyltransferase structure showed that, adjacent to the SAM-binding pocket, is a highly positively charged surface that could serve as an RNA binding site during cap methylations. Biochemical and mutagenesis analyses show that the N-7 and 2'-O cap methylations require distinct buffer conditions and different side chains within the K{sub 61}-D{sub 146}-K{sub 182}-E{sub 218} motif, suggesting that the two reactions use different mechanisms. In the context of complete virus, defects in both methylations are lethal to WNV; however, viruses defective solely in 2'-O methylation are attenuated and can protect mice from later wild-type WNV challenge. The results demonstrate that the N-7 methylation activity is essential for the WNV life cycle and, thus, methyltransferase represents a novel target for flavivirus therapy.« less

Preliminary characterization of (nucleoside-2′-O-)-methyltransferase crystals from Meaban and Yokose flaviviruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mastrangelo, Eloise; Bollati, Michela; Milani, Mario

2006-08-01

Two methyltransferases from flaviviruses (Meaban and Yokose viruses) have been overexpressed and crystallized. Diffraction data and characterization of the two crystal forms are presented, together with a preliminary molecular-replacement solution for both enzymes. Viral methyltranferases (MTase) are involved in the third step of the mRNA-capping process, transferring a methyl group from S-adenosyl-l-methionine (SAM) to the capped mRNA. MTases are classified into two groups: (guanine-N7)-methyltransferases (N7MTases), which add a methyl group onto the N7 atom of guanine, and (nucleoside-2′-O-)-methyltransferases (2′OMTases), which add a methyl group to a ribose hydroxyl. The MTases of two flaviviruses, Meaban and Yokose viruses, have been overexpressed,more » purified and crystallized in complex with SAM. Characterization of the crystals together with details of preliminary X-ray diffraction data collection (at 2.8 and 2.7 Å resolution, respectively) are reported here. The sequence homology relative to Dengue virus 2′OMTase and the structural conservation of specific residues in the putative active sites suggest that both enzymes belong to the 2′OMTase subgroup.« less

Management of elderly patients with glioblastoma-multiforme-a systematic review.

PubMed

Almadani, Asmaa; Sanjay, Dixit; Chris, Rowland-Hill; Shailendra, Achawal; Chitoor, Rajaraman; Gerry, O'Reilly; Robin, Highley; Masood, Hussain; Louise, Baker; Lynne, Gill; Holly, Morris; Mohan, Hingorani

2018-03-09

The management of elderly patients with glioblastoma-multiforme (GBM) remains poorly defined with many experts in the past advocating best supportive care, in view of limited evidence on efficacy of more aggressive treatment protocols. There is randomised evidence (NORDIC and NA-O8 studies) to support the use of surgery followed by adjuvant monotherapy with either radiotherapy (RT) using hypofractionated regimes (e.g. 36 Gy in 6 fractions OR 40 Gy in 15 fractions) or chemotherapy with temozolomide (TMZ) in patients expressing methylation of promoter for O 6 -methylguanine-DNA methyltransferase enzyme. However, the role of combined-modality therapy involving the use of combined RT and TMZ protocols has remained controversial with data from the EORTC (European Organisation for Research and Treatment of Cancer)-NCIC (National Cancer Institute of Canada) studies indicating that patients more than 65 years of age may not benefit significantly from combining standard RT fractionation using 60 Gy in 30 fractions with concurrent and adjuvant TMZ. More recently, randomised data has emerged on combining hypofractionated RT with concurrent and adjuvant TMZ. We provide a comprehensive review of literature with the aim of defining an evidence-based algorithm for management of elderly glioblastoma-multiforme population.

Characterization of Zea mays endosperm C-24 sterol methyltransferase: one of two types of sterol methyltransferase in higher plants.

PubMed

Grebenok, R J; Galbraith, D W; Penna, D D

1997-08-01

We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GALA regulation, the Z. mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide. Complementation was both plasmid-dependent and galactose-inducible. The Z. mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs). Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.

Identification of white campion (Silene latifolia) guaiacol O-methyltransferase involved in the biosynthesis of veratrole, a key volatile for pollinator attraction

PubMed Central

2012-01-01

Background Silene latifolia and its pollinator, the noctuid moth Hadena bicruris, represent an open nursery pollination system wherein floral volatiles, especially veratrole (1, 2-dimethoxybenzene), lilac aldehydes, and phenylacetaldehyde are of key importance for floral signaling. Despite the important role of floral scent in ensuring reproductive success in S. latifolia, the molecular basis of scent biosynthesis in this species has not yet been investigated. Results We isolated two full-length cDNAs from S. latifolia that show similarity to rose orcinol O-methyltransferase. Biochemical analysis showed that both S. latifolia guaiacol O-methyltransferase1 (SlGOMT1) &S. latifolia guaiacol O-methyltransferase2 (SlGOMT2) encode proteins that catalyze the methylation of guaiacol to form veratrole. A large Km value difference between SlGOMT1 (~10 μM) and SlGOMT2 (~501 μM) resulted that SlGOMT1 is 31-fold more catalytically efficient than SlGOMT2. qRT-PCR expression analysis showed that the SlGOMT genes are specifically expressed in flowers and male S. latifolia flowers had 3- to 4-folds higher level of GOMT gene transcripts than female flower tissues. Two related cDNAs, S. dioica O-methyltransferase1 (SdOMT1) and S. dioica O-methyltransferase2 (SdOMT2), were also obtained from the sister species Silene dioica, but the proteins they encode did not methylate guaiacol, consistent with the lack of veratrole emission in the flowers of this species. Our evolutionary analysis uncovered that SlGOMT1 and SlGOMT2 genes evolved under positive selection, whereas SdOMT1 and SdOMT2 genes show no evidence for selection. Conclusions Altogether, we report the identification and functional characterization of the gene, SlGOMT1 that efficiently catalyzes veratrole formation, whereas another copy of this gene with only one amino acid difference, SlGOMT2 was found to be less efficient for veratrole synthesis in S. latifolia. PMID:22937972

Radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culman, J.; Torda, T.; Weise, V.K.

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-(methyl-/sup 3/H) adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments ofmore » other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.« less

Dose finding and O6-alkylguanine-DNA alkyltransferase study of cisplatin combined with temozolomide in paediatric solid malignancies

PubMed Central

Geoerger, B; Vassal, G; Doz, F; O'Quigley, J; Wartelle, M; Watson, A J; Raquin, M-A; Frappaz, D; Chastagner, P; Gentet, J-C; Rubie, H; Couanet, D; Geoffray, A; Djafari, L; Margison, G P; Pein, F

2005-01-01

Cisplatin may have additive activity with temozolomide due to ablation of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (MGMT). This phase I/II study determined recommended combination doses using the Continual Reassessment Method, toxicities and antitumour activity in paediatric patients, and evaluated MGMT in peripheral blood mononuclear cells (PBMCs) in order to correlate with haematological toxicity. In total, 39 patients with refractory or recurrent solid tumours (median age ∼13 years; 14 pretreated with high-dose chemotherapy, craniospinal irradiation, or having bone marrow involvement) were treated with cisplatin, followed the next day by oral temozolomide for 5 days every 4 weeks at dose levels 80 mg m−2/150 mg m−2 day−1, 80/200, and 100/200, respectively. A total of 38 patients receiving 113 cycles (median 2, range 1–7) were evaluable for toxicity. Dose-limiting toxicity was haematological in all but one case. Treatment-related toxicities were thrombocytopenia, neutropenia, nausea-vomiting, asthenia. Hearing loss was experienced in five patients with prior irradiation to the brain stem or posterior fossa. Partial responses were observed in two malignant glioma, one brain stem glioma, and two neuroblastoma. Median MGMT activity in PBMCs decreased after 5 days of temozolomide treatment: low MGMT activity correlated with increased severity of thrombocytopenia. Cisplatin–temozolomide combinations are well tolerated without additional toxicity to single-agent treatments; the recommended phase II dosage is 80 mg m−2 cisplatin and 150 mg m−2 × 5 temozolomide in heavily treated, and 200 mg m−2 × 5 temozolomide in less-heavily pretreated children. PMID:16136028

Resistance of Salmonella typhimurium TA 1535 to O6-guanine methylation and mutagenesis induced by low doses of N-methyl-N'-nitro-N-nitrosoguanidine: an apparent constitutive repair activity.

PubMed

Guttenplan, J B; Milstein, S

1982-01-01

Salmonella tester strains which are reverted by base-pair substitution mutagens are relatively insensitive to the mutagenic effects of N-methyl-N-nitroso compounds. One reason for this insensitivity is the ability of these strains to withstand low doses of these compounds before they become sensitive to their mutagenic effects. In this report it is shown that mutagenesis induced by treatment of Salmonella typhimurium TA 1535 with N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) in buffer is biphasic with a low sensitivity range at low doses where little mutagenesis occurs, followed by a high sensitivity range whose onset begins after an apparent threshold dose has been exceeded. levels of O6-methylguanine (O6-MeG) in the DNA extracted from the bacteria follow a similar dose-response curve suggesting a dependency of mutagenesis on O6-MeG. In contrast, levels of 7-methylguanine (7-MeG) in the DNA increase linearly with dose. O6-MeG was undetectable at the lowest dose of MNNG whereas 7-MeG was readily detectable. Although such resistance to O6-alkylation has been demonstrated in MNNG- pretreated (adapted) E. coli, it has not been reported in unpretreated cells. Then isolated DNA was treated with MNNG a linear dose-response in the generation of O6-MeG was observed. The lack of O6-MeG in DNA isolated from MNNG treated cells after low doses is attributed to a saturable, constitutive repair activity in the bacteria. An attempt to observe the removal of O6-MeG from the bacteria after exposure to a short challenge dose of N-nitroso-N-methylurea (NMU) followed by a subsequent incubation in buffer was unsuccessful, probably because all the repair occurred within the time necessary to treat and lyse the cells.

Can the prognosis of individual patients with glioblastoma be predicted using an online calculator?

PubMed Central

Parks, Christopher; Heald, James; Hall, Gregory; Kamaly-Asl, Ian

2013-01-01

Background In an exploratory subanalysis of the European Organisation for Research and Treatment of Cancer and National Cancer Institute of Canada (EORTC/NCIC) trial data, Gorlia et al. identified a variety of factors that were predictive of overall survival, including therapy administered, age, extent of surgery, mini-mental score, administration of corticosteroids, World Health Organization (WHO) performance status, and O-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. Gorlia et al. developed 3 nomograms, each intended to predict the survival times of patients with newly diagnosed glioblastoma on the basis of individual-specific combinations of prognostic factors. These are available online as a “GBM Calculator” and are intended for use in patient counseling. This study is an external validation of this calculator. Method One hundred eighty-seven patients from 2 UK neurosurgical units who had histologically confirmed glioblastoma (WHO grade IV) had their information at diagnosis entered into the GBM calculator. A record was made of the actual and predicted median survival time for each patient. Statistical analysis was performed to assess the accuracy, precision, correlation, and discrimination of the calculator. Results The calculator gives both inaccurate and imprecise predictions. Only 23% of predictions were within 25% of the actual survival, and the percentage bias is 140% in our series. The coefficient of variance is 76%, where a smaller percentage would indicate greater precision. There is only a weak positive correlation between the predicted and actual survival among patients (R2 of 0.07). Discrimination is inadequate as measured by a C-index of 0.62. Conclusions The authors would not recommend the use of this tool in patient counseling. If departments were considering its use, we would advise that a similar validating exercise be undertaken. PMID:23543729

Molecular cloning, characterization and expression of the caffeic acid O-methyltransferase (COMT) ortholog from kenaf (Hibiscus cannabinus)

USDA-ARS?s Scientific Manuscript database

We cloned the full-length of the gene putatively encoding caffeic acid O-methyltransferase (COMT) from kenaf (Hibiscus cannabinus L.) using degenerate primers and the RACE (rapid amplification of cDNA ends) method. Kenaf is an herbaceous and rapidly growing dicotyledonous plant with great potential ...

Comparison of Promoter Hypermethylation Pattern in Salivary Rinses Collected with and without an Exfoliating Brush from Patients with HNSCC

PubMed Central

Sun, Wenyue; Zaboli, David; Liu, Yan; Arnaoutakis, Demetri; Khan, Tanbir; Wang, Hao; Koch, Wayne; Khan, Zubair; Califano, Joseph A.

2012-01-01

Background Salivary rinses have been recently proposed as a valuable resource for the development of epigenetic biomarkers for detection and monitoring of head and neck squamous cell carcinoma (HNSCC). Both salivary rinses collected with and without an exfoliating brush from patients with HNSCC are used in detection of promoter hypermethylation, yet their correlation of promoter hypermethylation has not been evaluated. This study was to evaluate the concordance of promoter hypermethylation between salivary rinses collected with and without an exfoliating brush from patients with HNSCC. Methodolgy 57 paired salivary rinses collected with or without an exfoliating brush from identical HNSCC patients were evaluated for promoter hypermethylation status using Quantitative Methylation-Specific PCR. Target tumor suppressor gene promoter regions were selected based on our previous studies describing a panel for HNSCC screening and surveillance, including P16, CCNA1, DCC, TIMP3, MGMT, DAPK and MINT31. Principal Findings In salivary rinses collected with and without brush, frequent methylation was detected in P16 (8.8% vs. 5.2%), CCNA1 (26.3% vs. 22.8%), DCC (33.3% vs. 29.8%), TIMP3 (31.6% vs. 36.8%), MGMT (29.8% vs. 38.6%), DAPK (14.0% vs. 19.2%), and MINT31 (10.5% vs. 8.8%). Spearman's rank correlation coefficient showed a positive correlation between salivary rinses collected with and without brush for P16 (ρ = 0.79), CCNA1 (ρ = 0.61), DCC (ρ = 0.58), TIMP3 (ρ = 0.10), MGMT (ρ = 0.70), DAPK (ρ = 0.51) and MINT31 (ρ = 0.72) (P<0.01). The percent agreement of promoter methylation between salivary rinses with brush and without brush were 96.5% for P16, 82.5% for CCNA1, 78.9% for DCC, 59.7% for TIMP3, 84.2% for MGMT, 84.2% for DAPK, and 94.7% for MINT31. Conclusions Our study demonstrated strong correlations of gene promoter hypermethylation between salivary rinses collected with and without an exfoliating brush. Salivary rinse collection

Conservation and functional importance of carbon-oxygen hydrogen bonding in AdoMet-dependent methyltransferases.

PubMed

Horowitz, Scott; Dirk, Lynnette M A; Yesselman, Joseph D; Nimtz, Jennifer S; Adhikari, Upendra; Mehl, Ryan A; Scheiner, Steve; Houtz, Robert L; Al-Hashimi, Hashim M; Trievel, Raymond C

2013-10-16

S-adenosylmethionine (AdoMet)-based methylation is integral to metabolism and signaling. AdoMet-dependent methyltransferases belong to multiple distinct classes and share a catalytic mechanism that arose through convergent evolution; however, fundamental determinants underlying this shared methyl transfer mechanism remain undefined. A survey of high-resolution crystal structures reveals that unconventional carbon-oxygen (CH···O) hydrogen bonds coordinate the AdoMet methyl group in different methyltransferases irrespective of their class, active site structure, or cofactor binding conformation. Corroborating these observations, quantum chemistry calculations demonstrate that these charged interactions formed by the AdoMet sulfonium cation are stronger than typical CH···O hydrogen bonds. Biochemical and structural studies using a model lysine methyltransferase and an active site mutant that abolishes CH···O hydrogen bonding to AdoMet illustrate that these interactions are important for high-affinity AdoMet binding and transition-state stabilization. Further, crystallographic and NMR dynamics experiments of the wild-type enzyme demonstrate that the CH···O hydrogen bonds constrain the motion of the AdoMet methyl group, potentially facilitating its alignment during catalysis. Collectively, the experimental findings with the model methyltransferase and structural survey imply that methyl CH···O hydrogen bonding represents a convergent evolutionary feature of AdoMet-dependent methyltransferases, mediating a universal mechanism for methyl transfer.

Cytotoxic Effects of Temozolomide and Radiation are Additive- and Schedule-Dependent

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chalmers, Anthony J., E-mail: a.j.chalmers@sussex.ac.u; Genome Damage and Stability Centre, University of Sussex, Falmer; Ruff, Elliot M.

2009-12-01

Purpose: Despite aggressive therapy comprising radical radiation and temozolomide (TMZ) chemotherapy, the prognosis for patients with glioblastoma multiforme (GBM) remains poor, particularly if tumors express O{sup 6}-methylguanine-DNA-methyltransferase (MGMT). The interactions between radiation and TMZ remain unclear and have important implications for scheduling and for developing strategies to improve outcomes. Methods and Materials: Factors determining the effects of combination therapy on clonogenic survival, cell-cycle checkpoint signaling and DNA repair were investigated in four human glioma cell lines (T98G, U373-MG, UVW, U87-MG). Results: Combining TMZ and radiation yielded additive cytotoxicity, but only when TMZ was delivered 72 h before radiation. Radiosensitization wasmore » not observed. TMZ induced G2/M cell-cycle arrest at 48-72 h, coincident with phosphorylation of Chk1 and Chk2. Additive G2/M arrest and Chk1/Chk2 phosphorylation was only observed when TMZ preceded radiation by 72 h. The ataxia-telangiectasia mutated (ATM) inhibitor KU-55933 increased radiation sensitivity and delayed repair of radiation-induced DNA breaks, but did not influence TMZ effects. The multiple kinase inhibitor caffeine enhanced the cytotoxicity of chemoradiation and exacerbated DNA damage. Conclusions: TMZ is not a radiosensitizing agent but yields additive cytotoxicity in combination with radiation. Our data indicate that TMZ treatment should commence at least 3 days before radiation to achieve maximum benefit. Activation of G2/M checkpoint signaling by TMZ and radiation has a cytoprotective effect that can be overcome by dual inhibition of ATM and ATR. More specific inhibition of checkpoint signaling will be required to increase treatment efficacy without exacerbating toxicity.« less

Fat Content and Nitrite-Curing Influence the Formation of Oxidation Products and NOC-Specific DNA Adducts during In Vitro Digestion of Meat

PubMed Central

Van Hecke, Thomas; Vossen, Els; Vanden Bussche, Julie; Raes, Katleen; Vanhaecke, Lynn; De Smet, Stefaan

2014-01-01

The effects of fat content and nitrite-curing of pork were investigated on the formation of cytotoxic and genotoxic lipid oxidation products (malondialdehyde, 4-hydroxy-2-nonenal, volatile simple aldehydes), protein oxidation products (protein carbonyl compounds) and NOC-specific DNA adducts (O6-carboxy-methylguanine) during in vitro digestion. The formation of these products during digestion is suggested to be responsible for the association between red meat and processed meat consumption and colorectal cancer risk. Digestion of uncured pork to which fat was added (total fat content 5 or 20%), resulted in significantly higher lipid and protein oxidation in the mimicked duodenal and colonic fluids, compared to digestion of pork without added fat (1% fat). A higher fat content also significantly favored the formation of O6-carboxy-methylguanine in the colon. Nitrite-curing of meat resulted in significantly lower lipid and protein oxidation before and after digestion, while an inconsistent effect on the formation of O6-carboxy-methylguanine was observed. The presented results demonstrate that haem-Fe is not solely responsible for oxidation and nitrosation reactions throughout an in vitro digestion approach but its effect is promoted by a higher fat content in meat. PMID:24978825

The translesion polymerase Rev3L in the tolerance of alkylating anticancer drugs.

PubMed

Roos, Wynand Paul; Tsaalbi-Shtylik, Anastasia; Tsaryk, Roman; Güvercin, Fatma; de Wind, Niels; Kaina, Bernd

2009-10-01

Temozolomide and fotemustine, representing methylating and chloroethylating agents, respectively, are used in the treatment of glioma and malignant melanoma. Because chemoresistance of these tumors is a common phenomenon, identification of the underlying mechanisms is needed. Here we show that Rev3L, the catalytic subunit of the translesion DNA polymerase zeta, mediates resistance to both temozolomide and fotemustine. Rev3L knockout cells are hypersensitive to both agents. It is remarkable that cells heterozygous for Rev3L showed an intermediate sensitivity. Rev3L is not involved in the tolerance of the toxic O6-methylguanine lesion. However, a possible role of Rev3L in the tolerance of O6-chloroethylguanine or the subsequently formed N1-guanine-N3-cytosine interstrand cross-link is shown. Rev3L had no influence on base excision repair (BER) of the N-alkylation lesions but is very likely to be involved in the tolerance of N-alkylations or apurinic/apyrimidinic sites originating from them. We also show that Rev3L exerts its protective effect in replicating cells and that loss of Rev3L leads to a significant increase in DNA double-strand breaks after temozolomide and fotemustine treatment. These data show that Rev3L contributes to temozolomide and fotemustine resistance, thus acting in concert with O6-methylguanine-DNA methyltransferase, BER, mismatch repair, and double-strand break repair in defense against simple alkylating anticancer drugs.

Rational design of a live attenuated dengue vaccine: 2'-o-methyltransferase mutants are highly attenuated and immunogenic in mice and macaques.

PubMed

Züst, Roland; Dong, Hongping; Li, Xiao-Feng; Chang, David C; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R; Ellis, Esther M; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2'-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2'-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2'-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response.

Cloning and Functional Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.)

PubMed Central

Niu, Gai-Li; Gou, Wei; Han, Xiang-Long; Qin, Cheng; Zhang, Li-Xin; Ashraf, Muhammad

2018-01-01

Betaine, a non-toxic osmoprotectant, is believed to accumulate considerably in plants under stress conditions to maintain the osmotic pressure and promote a variety of processes involved in growth and development. Phosphoethanolamine N-methyltransferase (PEAMT), a key enzyme for betaine synthesis, is reported to be regulated by its upstream promoter. In the present investigation, by using the transgenic approach, a 1048 bp long promoter region of ZmPEAMT gene from Zea mays was cloned and functionally characterized in tobacco. Computational analysis affirmed the existence of abiotic stress responsive cis-elements like ABRE, MYC, HST, LST etc., as well as pathogen, wound and phytohormone responsive motifs. For transformation in tobacco, four 5′-deletion constructs of 826 bp (P2), 642 bp (P3), 428 bp (P4) and 245 bp (P5) were constructed from the 1048 bp (P1) promoter fragment. The transgenic plants generated through a single event exhibited a promising expression of GUS reporter protein in the leaf tissues of treated with salt, drought, oxidative and cold stress as well as control plants. The GUS expression level progressively reduced from P1 to P5 in the leaf tissues, whereas a maximal expression was observed with the P3 construct in the leaves of control plants. The expression of GUS was noted to be higher in the leaves of osmotically- or salt-treated transgenic plants than that in the untreated (control) plants. An effective expression of GUS in the transgenic plants manifests that this promoter can be employed for both stress-inducible and constitutive expression of gene(s). Due to this characteristic, this potential promoter can be effectively used for genetic engineering of several crops. PMID:29316727

Short peptides derived from the interaction domain of SARS coronavirus nonstructural protein nsp10 can suppress the 2'-O-methyltransferase activity of nsp10/nsp16 complex.

PubMed

Ke, Min; Chen, Yu; Wu, Andong; Sun, Ying; Su, Ceyang; Wu, Hao; Jin, Xu; Tao, Jiali; Wang, Yi; Ma, Xiao; Pan, Ji-An; Guo, Deyin

2012-08-01

Coronaviruses are the etiological agents of respiratory and enteric diseases in humans and livestock, exemplified by the life-threatening severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV). However, effective means for combating coronaviruses are still lacking. The interaction between nonstructural protein (nsp) 10 and nsp16 has been demonstrated and the crystal structure of SARS-CoV nsp16/10 complex has been revealed. As nsp10 acts as an essential trigger to activate the 2'-O-methyltransferase activity of nsp16, short peptides derived from nsp10 may have inhibitory effect on viral 2'-O-methyltransferase activity. In this study, we revealed that the domain of aa 65-107 of nsp10 was sufficient for its interaction with nsp16 and the region of aa 42-120 in nsp10, which is larger than the interaction domain, was needed for stimulating the nsp16 2'-O-methyltransferase activity. We further showed that two short peptides derived from the interaction domain of nsp10 could inhibit the 2'-O-methyltransferase activity of SARS-CoV nsp16/10 complex, thus providing a novel strategy and proof-of-principle study for developing peptide inhibitors against SARS-CoV. Copyright © 2012 Elsevier B.V. All rights reserved.

Cotransduction with MGMT and Ubiquitous or Erythroid-Specific GFP Lentiviruses Allows Enrichment of Dual-Positive Hematopoietic Progenitor Cells In Vivo

PubMed Central

Roth, Justin C.; Ismail, Mourad; Reese, Jane S.; Lingas, Karen T.; Ferrari, Giuliana; Gerson, Stanton L.

2012-01-01

The P140K point mutant of MGMT allows robust hematopoietic stem cell (HSC) enrichment in vivo. Thus, dual-gene vectors that couple MGMT and therapeutic gene expression have allowed enrichment of gene-corrected HSCs in animal models. However, expression levels from dual-gene vectors are often reduced for one or both genes. Further, it may be desirable to express selection and therapeutic genes at distinct stages of cell differentiation. In this regard, we evaluated whether hematopoietic cells could be efficiently cotransduced using low MOIs of two separate single-gene lentiviruses, including MGMT for dual-positive cell enrichment. Cotransduction efficiencies were evaluated using a range of MGMT : GFP virus ratios, MOIs, and selection stringencies in vitro. Cotransduction was optimal when equal proportions of each virus were used, but low MGMT : GFP virus ratios resulted in the highest proportion of dual-positive cells after selection. This strategy was then evaluated in murine models for in vivo selection of HSCs cotransduced with a ubiquitous MGMT expression vector and an erythroid-specific GFP vector. Although the MGMT and GFP expression percentages were variable among engrafted recipients, drug selection enriched MGMT-positive leukocyte and GFP-positive erythroid cell populations. These data demonstrate cotransduction as a mean to rapidly enrich and evaluate therapeutic lentivectors in vivo. PMID:22888445

Nuclear carbonic anhydrase 6B associates with PRMT5 to epigenetically promote IL-12 expression in innate response.

PubMed

Xu, Jia; Xu, Xiaoqing; Wang, Bingjing; Ma, Yuanwu; Zhang, Lianfeng; Xu, Henan; Hu, Ye; Wu, Jiacheng; Cao, Xuetao

2017-08-08

Interleukin-12 (IL-12) is critical for induction of protective immunity against intracellular bacterial infection. However, the mechanisms for efficient induction of IL-12 in innate response remain poorly understood. Here we report that the B type of carbonic anhydrase 6 ( Car6-b , which encoded CA-VI B) is essential for host defense against Listeria monocytogenes (LM) infection by epigenetically promoting IL-12 expression independent of its carbonic anhydrase activity. Deficiency of Car6-b attenuated IL-12 production upon LM infection both in vitro and in vivo. Car6 -/- mice were more susceptible to LM infection with less production of IL-12. Mechanistically, the nuclear localized CA-VI B selectively promotes IL-12 expression by interaction with protein arginine N -methyltransferase 5 (PRMT5), which reduces symmetric dimethylation of histone H3 arginine 8 modification (H3R8me2s) at Il12 promoters to facilitate chromatin accessibility, selectively enhancing c-Rel binding to the Il12b promoter. Our findings add insights to the epigenetic regulation of IL-12 induction in innate immunity.

Synthesis of Lysine Methyltransferase Inhibitors

NASA Astrophysics Data System (ADS)

Ye, Tao; Hui, Chunngai

2015-07-01

Lysine methyltransferase which catalyze methylation of histone and nonhistone proteins, play a crucial role in diverse biological processes and has emerged as a promising target for the development of various human diseases, including cancer, inflammation, and psychiatric disorders. However, inhibiting Lysine methyltransferases selectively has presented many challenges to medicinal chemists. During the past decade, lysine methyltransferase inhibitors covering many different structural classes have been designed and developed. In this review, we describe the development of selective, small-molecule inhibitors of lysine methyltransferases with an emphasis on their discovery and chemical synthesis. We highlight the current state of lysine methyltransferase inhibitors and discuss future directions and opportunities for lysine methyltransferase inhibitor discovery.

Associations between arsenic (+3 oxidation state) methyltransferase (AS3MT) and N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) polymorphisms, arsenic metabolism, and cancer risk in a chilean population.

PubMed

de la Rosa, Rosemarie; Steinmaus, Craig; Akers, Nicholas K; Conde, Lucia; Ferreccio, Catterina; Kalman, David; Zhang, Kevin R; Skibola, Christine F; Smith, Allan H; Zhang, Luoping; Smith, Martyn T

2017-07-01

Inter-individual differences in arsenic metabolism have been linked to arsenic-related disease risks. Arsenic (+3) methyltransferase (AS3MT) is the primary enzyme involved in arsenic metabolism, and we previously demonstrated in vitro that N-6 adenine-specific DNA methyltransferase 1 (N6AMT1) also methylates the toxic inorganic arsenic (iAs) metabolite, monomethylarsonous acid (MMA), to the less toxic dimethylarsonic acid (DMA). Here, we evaluated whether AS3MT and N6AMT1 gene polymorphisms alter arsenic methylation and impact iAs-related cancer risks. We assessed AS3MT and N6AMT1 polymorphisms and urinary arsenic metabolites (%iAs, %MMA, %DMA) in 722 subjects from an arsenic-cancer case-control study in a uniquely exposed area in northern Chile. Polymorphisms were genotyped using a custom designed multiplex, ligation-dependent probe amplification (MLPA) assay for 6 AS3MT SNPs and 14 tag SNPs in the N6AMT1 gene. We found several AS3MT polymorphisms associated with both urinary arsenic metabolite profiles and cancer risk. For example, compared to wildtypes, individuals carrying minor alleles in AS3MT rs3740393 had lower %MMA (mean difference = -1.9%, 95% CI: -3.3, -0.4), higher %DMA (mean difference = 4.0%, 95% CI: 1.5, 6.5), and lower odds ratios for bladder (OR = 0.3; 95% CI: 0.1-0.6) and lung cancer (OR = 0.6; 95% CI: 0.2-1.1). Evidence of interaction was also observed for both lung and bladder cancer between these polymorphisms and elevated historical arsenic exposures. Clear associations were not seen for N6AMT1. These results are the first to demonstrate a direct association between AS3MT polymorphisms and arsenic-related internal cancer risk. This research could help identify subpopulations that are particularly vulnerable to arsenic-related disease. Environ. Mol. Mutagen. 58:411-422, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Specialized (iso)eugenol-4-O-methyltransferases (s-IEMTs) and methods of making and using the same

DOEpatents

Liu, Chang-Jun; Cai, Yuanheng

2017-01-31

Specialized (iso)eugenol 4-O-methyltransferase (s-IEMT) enzymes having increased capacity for methylation of monolignols are disclosed. The s-IEMTs have unique activity favoring methylation of coniferyl alcohol versus sinapyl alcohol. Various s-IEMTs methylate ferulic acid. Means for producing the various s-IEMTs are provided. The s-IEMTs are useful for modification of lignin content and production of aromatic compounds.

Small Molecule Inhibitors That Selectively Block Dengue Virus Methyltransferase*

PubMed Central

Lim, Siew Pheng; Sonntag, Louis Sebastian; Noble, Christian; Nilar, Shahul H.; Ng, Ru Hui; Zou, Gang; Monaghan, Paul; Chung, Ka Yan; Dong, Hongping; Liu, Boping; Bodenreider, Christophe; Lee, Gladys; Ding, Mei; Chan, Wai Ling; Wang, Gang; Jian, Yap Li; Chao, Alexander Theodore; Lescar, Julien; Yin, Zheng; Vedananda, T. R.; Keller, Thomas H.; Shi, Pei-Yong

2011-01-01

Crystal structure analysis of Flavivirus methyltransferases uncovered a flavivirus-conserved cavity located next to the binding site for its cofactor, S-adenosyl-methionine (SAM). Chemical derivatization of S-adenosyl-homocysteine (SAH), the product inhibitor of the methylation reaction, with substituents that extend into the identified cavity, generated inhibitors that showed improved and selective activity against dengue virus methyltransferase (MTase), but not related human enzymes. Crystal structure of dengue virus MTase with a bound SAH derivative revealed that its N6-substituent bound in this cavity and induced conformation changes in residues lining the pocket. These findings demonstrate that one of the major hurdles for the development of methyltransferase-based therapeutics, namely selectivity for disease-related methyltransferases, can be overcome. PMID:21147775

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

PubMed Central

Li, Laigeng; Popko, Jacqueline L.; Zhang, Xing-Hai; Osakabe, Keishi; Tsai, Chung-Jui; Joshi, Chandrashekhar P.; Chiang, Vincent L.

1997-01-01

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem. PMID:9144260

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine.

PubMed

Li, L; Popko, J L; Zhang, X H; Osakabe, K; Tsai, C J; Joshi, C P; Chiang, V L

1997-05-13

S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Enhanced detoxification and degradation of herbicide atrazine by a group of O-methyltransferases in rice.

PubMed

Lu, Yi Chen; Luo, Fang; Pu, Zhong Ji; Zhang, Shuang; Huang, Meng Tian; Yang, Hong

2016-12-01

Atrazine (ATR) as a toxic herbicide has become one of the seriously environmental contaminants worldwide due to its long-term intensive use in crop production. This study identified novel methyltransferases (MTs) involved in detoxification and degradation of ATR residues in rice plants. From a subset of MTs differentially expressed in ATR-exposed rice, forty-four O-methyltransferase genes were investigated. Total activities were significantly enhanced by ATR in rice tissues. To prove detoxifying capacity of the MTs in rice plants, two rice O-MTs (LOC_Os04g09604 and LOC_Os11g15040) were selected and transformed into yeast cells (Pichia pastoris X-33). The positive transformants accumulated less ATR and showed less toxicity. Using UPLC-TOF-MS/MS, ATR-degraded products in rice and yeast cells were characterized. A novel O-methylated-modified metabolite (atraton) and six other ATR-derivatives were detected. The topological interaction between LOC_Os04g09604 enzyme and its substrate was specially analyzed by homology modeling programs, which was well confirmed by the molecular docking analysis. The significance of the study is to provide a better understanding of mechanisms for the specific detoxification and degradation of ATR residues in rice growing in environmentally relevant ATR-contaminated soils and may hold a potential engineering perspective for generating ATR-resistant rice that helps to minimize ATR residues in crops. Copyright © 2016 Elsevier Ltd. All rights reserved.

Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.).

PubMed

Brenner, Everton A; Zein, Imad; Chen, Yongsheng; Andersen, Jeppe R; Wenzel, Gerhard; Ouzunova, Milena; Eder, Joachim; Darnhofer, Birte; Frei, Uschi; Barrière, Yves; Lübberstedt, Thomas

2010-02-12

OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Variation in genomic sequences coding for COMT, CCoAOMT1, and CCoAOMT2 was analyzed in relation to stover cell wall digestibility for a panel of 40 European forage maize inbred lines, and re-analyzed for a panel of 34 lines from a published French study. Different methodologies for association analysis were performed and compared. Across association methodologies, a total number of 25, 12, 1, 6 COMT polymorphic sites were significantly associated with DNDF, OMD, NDF, and WSC, respectively. Association analysis for CCoAOMT1 and CCoAOMT2 identified substantially fewer polymorphic sites (3 and 2, respectively) associated with the investigated traits. Our re-analysis on the 34 lines from a published French dataset identified 14 polymorphic sites significantly associated with cell wall digestibility, two of them were consistent with our study. Promising polymorphisms putatively causally associated with variability of cell wall digestibility were inferred from the total number of significantly associated SNPs/Indels. Several polymorphic sites for three O-methyltransferase loci were associated with stover cell wall digestibility. All three tested genes seem to be involved in controlling DNDF, in particular COMT. Thus, considerable variation among Bm3 wildtype alleles can be exploited for improving cell-wall digestibility. Target sites for functional markers were identified enabling development of efficient marker-based selection strategies.

Polymorphisms in O-methyltransferase genes are associated with stover cell wall digestibility in European maize (Zea mays L.)

PubMed Central

2010-01-01

Background OMT (O-methyltransferase) genes are involved in lignin biosynthesis, which relates to stover cell wall digestibility. Reduced lignin content is an important determinant of both forage quality and ethanol conversion efficiency of maize stover. Results Variation in genomic sequences coding for COMT, CCoAOMT1, and CCoAOMT2 was analyzed in relation to stover cell wall digestibility for a panel of 40 European forage maize inbred lines, and re-analyzed for a panel of 34 lines from a published French study. Different methodologies for association analysis were performed and compared. Across association methodologies, a total number of 25, 12, 1, 6 COMT polymorphic sites were significantly associated with DNDF, OMD, NDF, and WSC, respectively. Association analysis for CCoAOMT1 and CCoAOMT2 identified substantially fewer polymorphic sites (3 and 2, respectively) associated with the investigated traits. Our re-analysis on the 34 lines from a published French dataset identified 14 polymorphic sites significantly associated with cell wall digestibility, two of them were consistent with our study. Promising polymorphisms putatively causally associated with variability of cell wall digestibility were inferred from the total number of significantly associated SNPs/Indels. Conclusions Several polymorphic sites for three O-methyltransferase loci were associated with stover cell wall digestibility. All three tested genes seem to be involved in controlling DNDF, in particular COMT. Thus, considerable variation among Bm3 wildtype alleles can be exploited for improving cell-wall digestibility. Target sites for functional markers were identified enabling development of efficient marker-based selection strategies. PMID:20152036

Melatonin production in Escherichia coli by dual expression of serotonin N-acetyltransferase and caffeic acid O-methyltransferase.

PubMed

Byeon, Yeong; Back, Kyoungwhan

2016-08-01

Melatonin is a well-known bioactive molecule produced in animals and plants and a well-studied natural compound. Two enzymatic steps are required for the biosynthesis of melatonin from serotonin. First, serotonin N-acetyltransferase (SNAT) catalyzes serotonin to N-acetylserotonin (NAS) followed by the action of N-acetylserotonin O-methyltransferase (ASMT), resulting in the synthesis of O-methylated NAS, also known as melatonin. Attempts to document melatonin production in Escherichia coli have been unsuccessful to date due to either low enzyme activity or inactive ASMT expression. Here, we employed caffeic acid O-methyltransferase (COMT) instead of ASMT, as COMT is a multifunctional enzyme that has ASMT activity as well. Among several combinations of dual expression cassettes, recombinant E. coli that expressed sheep SNAT with rice COMT produced a high quantity of melatonin, which was measured in a culture medium (1.46 mg/L in response to 1 mM serotonin). This level was several orders of magnitude higher than that produced in transgenic rice and tomato overexpressing sheep SNAT and ASMT, respectively. This heterologous expression system can be widely employed to screen various putative SNAT or ASMT genes from animals and plants as well as to overproduce melatonin in various useful microorganisms.

Modulation of O6-alkylating agent induced clastogenicity by enhanced DNA repair capacity of bone marrow cells.

PubMed

Chinnasamy, N; Fairbairn, L J; Laher, J; Willington, M A; Rafferty, J A

1998-08-07

The murine bone marrow micronucleus assay has been used to examine (1) the potentiation of fotemustine and streptozotocin induced-clastogenicity by the O6-alkylguanine-DNA alkyltransferase (ATase) inactivator O6-benzylguanine (O6-beG) and (2) the level of protection afforded against this potentiation by retrovirus-mediated expression of an O6-beG-resistant mutant of human ATase (haTPA/GA) in mouse bone marrow. Both fotemustine and streptozotocin induced significantly higher levels of micronucleated polychromatic erythrocytes (p < 0.001 for the highest doses studied) compared to those seen in vehicle-treated animals. The number of micronuclei produced by either agent was dramatically elevated by pretreatment with O6-beG (p < 0.001). Furthermore, in myeloablated mice reconstituted with bone marrow expressing the O6-beG-resistant hATPA/GA as a result of retroviral gene transfer, the frequency of micronucleus formation following exposure of mice to otherwise clastogenic doses of fotemustine or streptozotocin, in the presence of O6-beG, wash highly significantly reduced (p < 0.001 for both agents) relative to that in mock transduced controls. These data clearly implicate O6-chloroethyl- and O6-methylguanine as clastogenic lesions in vivo and establish ATase as a major protective mechanism operating to reduce the frequency of such damage. The potentiation of drug induced clastogenicity by O6-beG suggests that the clinical use of this inactivator in combination with O6-alkylating agents, could substantially increase the risk of therapy related malignancy. Nevertheless the use of hATPA/GA as a protective mechanism via gene therapy may overcome this risk.

Two transcriptional activators of N-acetylserotonin O-methyltransferase 2 and melatonin biosynthesis in cassava.

PubMed

Wei, Yunxie; Liu, Guoyin; Bai, Yujing; Xia, Feiyu; He, Chaozu; Shi, Haitao; Foyer, Christine

2017-10-13

Similar to the situation in animals, melatonin biosynthesis is regulated by four sequential enzymatic steps in plants. Although the melatonin synthesis genes have been identified in various plants, the upstream transcription factors of them remain unknown. In this study on cassava (Manihot esculenta), we found that MeWRKY79 and heat-shock transcription factor 20 (MeHsf20) targeted the W-box and the heat-stress elements (HSEs) in the promoter of N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively. The interaction between MeWRKY79, MeHsf20, and the MeASMT2 promoter was evidenced by the activation of promoter activity and chromatin immunoprecipitation (ChIP) in cassava protoplasts, and by an in vitro electrophoretic mobility shift assay (EMSA). The transcripts of MeWRKY79, MeHsf20, and MeASMT2 were all regulated by a 22-amino acid flagellin peptide (flg22) and by Xanthomonas axonopodis pv manihotis (Xam). In common with the phenotype of MeASMT2, transient expression of MeWRKY79 and MeHsf20 in Nicotiana benthamiana leaves conferred improved disease resistance. Through virus-induced gene silencing (VIGS) in cassava, we found that MeWRKY79- and MeHsf20-silenced plants showed lower transcripts of MeASMT2 and less accumulation of melatonin, which resulted in disease sensitivity that could be reversed by exogenous melatonin. Taken together, these results indicate that MeASMT2 is a target of MeWRKY79 and MeHsf20 in plant disease resistance. This study identifies novel upstream transcription factors of melatonin synthesis genes in cassava, thus extending our knowledge of the complex modulation of melatonin synthesis in plant defense. © The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Rational Design of a Live Attenuated Dengue Vaccine: 2′-O-Methyltransferase Mutants Are Highly Attenuated and Immunogenic in Mice and Macaques

PubMed Central

Chang, David C.; Zhang, Bo; Balakrishnan, Thavamalar; Toh, Ying-Xiu; Jiang, Tao; Li, Shi-Hua; Deng, Yong-Qiang; Ellis, Brett R.; Ellis, Esther M.; Poidinger, Michael; Zolezzi, Francesca; Qin, Cheng-Feng; Shi, Pei-Yong; Fink, Katja

2013-01-01

Dengue virus is transmitted by Aedes mosquitoes and infects at least 100 million people every year. Progressive urbanization in Asia and South-Central America and the geographic expansion of Aedes mosquito habitats have accelerated the global spread of dengue, resulting in a continuously increasing number of cases. A cost-effective, safe vaccine conferring protection with ideally a single injection could stop dengue transmission. Current vaccine candidates require several booster injections or do not provide protection against all four serotypes. Here we demonstrate that dengue virus mutants lacking 2′-O-methyltransferase activity are highly sensitive to type I IFN inhibition. The mutant viruses are attenuated in mice and rhesus monkeys and elicit a strong adaptive immune response. Monkeys immunized with a single dose of 2′-O-methyltransferase mutant virus showed 100% sero-conversion even when a dose as low as 1,000 plaque forming units was administrated. Animals were fully protected against a homologous challenge. Furthermore, mosquitoes feeding on blood containing the mutant virus were not infected, whereas those feeding on blood containing wild-type virus were infected and thus able to transmit it. These results show the potential of 2′-O-methyltransferase mutant virus as a safe, rationally designed dengue vaccine that restrains itself due to the increased susceptibility to the host's innate immune response. PMID:23935499

How Metal Substitution Affects the Enzymatic Activity of Catechol-O-Methyltransferase

PubMed Central

Sparta, Manuel; Alexandrova, Anastassia N.

2012-01-01

Catechol-O-methyltransferase (COMT) degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II)), and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II) with Ca(II) leads to a complete deactivation of COMT; Fe(II) is slightly less than potent Mg(II), and Fe(III) is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II) bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II), whereas Fe(III) is unable to promote catalysis. In the case of Ca(II), a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III) it is the electronic effect, whereas in the case of Ca(II) it is instead the effect of suboptimal protein structure. PMID:23056605

Catechol-O-Methyltransferase "Val[superscript 158]Met" Genotype, Parenting Practices and Adolescent Alcohol Use: Testing the Differential Susceptibility Hypothesis

ERIC Educational Resources Information Center

Laucht, Manfred; Blomeyer, Dorothea; Buchmann, Arlette F.; Treutlein, Jens; Schmidt, Martin H.; Esser, Gunter; Jennen-Steinmetz, Christine; Rietschel, Marcella; Zimmermann, Ulrich S.; Banaschewski, Tobias

2012-01-01

Background: Recently, first evidence has been reported for a gene-parenting interaction (G x E) with regard to adolescent alcohol use. The present investigation set out to extend this research using the catechol-O-methyltransferase ("COMT") "Val[superscript 158]Met" polymorphism as a genetic susceptibility factor. Moreover, the current study…

Site-specific bioalkylation of rapamycin by the RapM 16-O-methyltransferase.

PubMed

Law, Brian J C; Struck, Anna-Winona; Bennett, Matthew R; Wilkinson, Barrie; Micklefield, Jason

2015-05-01

The methylation of natural products by S -adenosyl methionine (AdoMet, also known as SAM)-dependent methyltransferase enzymes is a common tailoring step in many biosynthetic pathways. The introduction of methyl substituents can affect the biological and physicochemical properties of the secondary metabolites produced. Recently it has become apparent that some AdoMet-dependent methyltransferases exhibit promiscuity and will accept AdoMet analogues enabling the transfer of alternative alkyl groups. In this study we have characterised a methyltransferase, RapM, which is involved in the biosynthesis of the potent immunosuppressive agent rapamycin. We have shown that recombinant RapM regioselectively methylates the C16 hydroxyl group of desmethyl rapamycin precursors in vitro and is promiscuous in accepting alternative co-factors in addition to AdoMet. A coupled enzyme system was developed, including a mutant human enzyme methionine adenosyl transferase (MAT), along with RapM, which was used to prepare alkylated rapamycin derivatives (rapalogs) with alternative ethyl and allyl ether groups, derived from simple S -ethyl or S -allyl methionine analogues. There are two other methyltransferases RapI and RapQ which provide methyl substituents of rapamycin. Consequently, using the enzymatic approach described here, it should be possible to generate a diverse array of alkylated rapalogs, with altered properties, that would be difficult to obtain by traditional synthetic approaches.

The effect of 6-methylthiohexyl isothiocyanate isolated from Wasabia japonica (wasabi) on 4-(methylnitrosamino)-1-(3-pyridyl)-1-buatnone-induced lung tumorigenesis in mice.

PubMed

Yano, T; Yajima, S; Virgona, N; Yano, Y; Otani, S; Kumagai, H; Sakurai, H; Kishimoto, M; Ichikawa, T

2000-07-31

The present study was undertaken to estimate the effect of 6-methylthiohexyl isothiocyanate (6MHITC) isolated from Wasabia japonica (wasabi) pretreatment on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone(NNK)-induced lung tumorigenesis in mice. Pretreatment with 6MHITC for 4 consecutive days at a daily dose of 5 micromol significantly inhibited NNK-induced O(6)-methylguanine formation in lungs at 4 h after the injection. In conjugation with this inhibitory effect, 6MHITC suppressed the increase in proliferating nuclear cell antigen level as well as ornithine decarboxylase activity at a promotion stage of NNK-induced lung tumorigenesis. Finally, this treatment of 6MHITC suppressed the NNK-induced lung tumorigenesis in mice. These results suggest that 6MHITC inhibits the development of lung tumors in mice treated with NNK, due to the suppression of initiation stage.

Accelerated degradation of lignin by lignin peroxidase isozyme H8 (LiPH8) from Phanerochaete chrysosporium with engineered 4-O-methyltransferase from Clarkia breweri.

PubMed

Pham, Le Thanh Mai; Kim, Yong Hwan

2014-11-01

Free-hydroxyl phenolic units can decrease or even abort the catalytic activity of lignin peroxidase H8 during oxidation of veratryl alcohol and model lignin dimers, resulting in slow and inefficient lignin degradation. In this study we applied engineered 4-O-methyltransferase from Clarkia breweri to detoxify the inhibiting free-hydroxyl phenolic groups by converting them to methylated phenolic groups. The multistep, enzyme-catalyzed process that combines 4-O-methyltransferase and lignin peroxidase H8 suggested in this work can increase the efficiency of lignin-degradation. This study also suggests approaching the field of multi-enzyme in vitro systems to improve the understanding and development of plant biomass in biorefinery operations. Copyright © 2014 Elsevier Inc. All rights reserved.

DNA-binding mechanism of the Escherichia coli Ada O6-alkylguanine–DNA alkyltransferase

PubMed Central

Verdemato, Philip E.; Brannigan, James A.; Damblon, Christian; Zuccotto, Fabio; Moody, Peter C. E.; Lian, Lu-Yun

2000-01-01

The C-terminal domain of the Escherichia coli Ada protein (Ada-C) aids in the maintenance of genomic integrity by efficiently repairing pre-mutagenic O6-alkylguanine lesions in DNA. Structural and thermodynamic studies were carried out to obtain a model of the DNA-binding process. Nuclear magnetic resonance (NMR) studies map the DNA-binding site to helix 5, and a loop region (residues 151–160) which form the recognition helix and the ‘wing’ of a helix–turn–wing motif, respectively. The NMR data also suggest the absence of a large conformational change in the protein upon binding to DNA. Hence, an O6-methylguanine (O6meG) lesion would be inaccessible to active site nucleophile Cys146 if the modified base remained stacked within the DNA duplex. The experimentally determined DNA-binding face of Ada-C was used in combination with homology modelling, based on the catabolite activator protein, and the accepted base-flipping mechanism, to construct a model of how Ada-C binds to DNA in a productive manner. To complement the structural studies, thermodynamic data were obtained which demonstrate that binding to unmethylated DNA was entropically driven, whilst the demethylation reaction provoked an exothermic heat change. Methylation of Cys146 leads to a loss of structural integrity of the DNA-binding subdomain. PMID:11000262

Functional and Structural Analysis of Phenazine O-Methyltransferase LaPhzM from Lysobacter antibioticus OH13 and One-Pot Enzymatic Synthesis of the Antibiotic Myxin.

PubMed

Jiang, Jiasong; Guiza Beltran, Daisy; Schacht, Andrew; Wright, Stephen; Zhang, Limei; Du, Liangcheng

2018-04-20

Myxin is a well-known antibiotic that had been used for decades. It belongs to the phenazine natural products that exhibit various biological activities, which are often dictated by the decorating groups on the heteroaromatic three-ring system. The three rings of myxin carry a number of decorations, including an unusual aromatic N5, N10-dioxide. We previously showed that phenazine 1,6-dicarboxylic acid (PDC) is the direct precursor of myxin, and two redox enzymes (LaPhzS and LaPhzNO1) catalyze the decarboxylative hydroxylation and aromatic N-oxidations of PDC to produce iodinin (1.6-dihydroxy- N5, N10-dioxide phenazine). In this work, we identified the LaPhzM gene from Lysobacter antibioticus OH13 and demonstrated that LaPhzM encodes a SAM-dependent O-methyltransferase converting iodinin to myxin. The results further showed that LaPhzM is responsible for both monomethoxy and dimethoxy formation in all phenazine compounds isolated from strain OH13. LaPhzM exhibits relaxed substrate selectivity, catalyzing O-methylation of phenazines with non-, mono-, or di- N-oxide. In addition, we demonstrated a one-pot biosynthesis of myxin by in vitro reconstitution of the three phenazine-ring decorating enzymes. Finally, we determined the X-ray crystal structure of LaPhzM with a bound cofactor at 1.4 Å resolution. The structure provided molecular insights into the activity and selectivity of the first characterized phenazine O-methyltransferase. These results will facilitate future exploitation of the thousands of phenazines as new antibiotics through metabolic engineering and chemoenzymatic syntheses.

K-ras gene sequence effects on the formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-DNA adducts.

PubMed

Ziegel, Rebecca; Shallop, Anthony; Jones, Roger; Tretyakova, Natalia

2003-04-01

The tobacco specific pulmonary carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolically activated to electrophilic species that form methyl and pyridyloxobutyl adducts with genomic DNA, including O(6)-methylguanine, N7-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine. If not repaired, these lesions could lead to mutations and the initiation of cancer. Previous studies used ligation-mediated polymerase chain reaction (LMPCR) in combination with PAGE to examine the distribution of NNK-induced strand breaks and alkali labile lesions (e.g., N7-methylguanine) within gene sequences. However, LMPCR cannot be used to establish the distribution patterns of highly promutagenic O(6)-methylguanine and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts of NNK. We have developed methods based on stable isotope labeling HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI MS/MS) that enable us to accurately quantify NNK-induced adducts at defined sites within DNA sequences. In the present study, the formation of N7-methylguanine, O(6)-methylguanine, and O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine adducts at specific positions within a K-ras gene-derived double-stranded DNA sequence (5'-G(1)G(2)AG(3)CTG(4)G(5)TG(6)G(7)CG(8)TA G(9)G(10)C-3') was investigated following treatment with activated NNK metabolites. All three lesions preferentially formed at the second position of codon 12 (GGT), the major mutational hotspot for G-->A and G-->T base substitutions observed in smoking-induced lung tumors. Therefore, our data support the involvement of NNK and other tobacco specific nitrosamines in mutagenesis and carcinogenesis.

Molecular markers of carcinogenesis for risk stratification of individuals with colorectal polyps: a case-control study.

PubMed

Gupta, Samir; Sun, Han; Yi, Sang; Storm, Joy; Xiao, Guanghua; Balasubramanian, Bijal A; Zhang, Song; Ashfaq, Raheela; Rockey, Don C

2014-10-01

Risk stratification using number, size, and histology of colorectal adenomas is currently suboptimal for identifying patients at increased risk for future colorectal cancer. We hypothesized that molecular markers of carcinogenesis in adenomas, measured via immunohistochemistry, may help identify high-risk patients. To test this hypothesis, we conducted a retrospective, 1:1 matched case-control study (n = 216; 46% female) in which cases were patients with colorectal cancer and synchronous adenoma and controls were patients with adenoma but no colorectal cancer at baseline or within 5 years of follow-up. In phase I of analyses, we compared expression of molecular markers of carcinogenesis in case and control adenomas, blind to case status. In phase II of analyses, patients were randomly divided into independent training and validation groups to develop a model for predicting case status. We found that seven markers [p53, p21, Cox-2, β-catenin (BCAT), DNA-dependent protein kinase (DNApkcs), survivin, and O6-methylguanine-DNA methyltransferase (MGMT)] were significantly associated with case status on unadjusted analyses, as well as analyses adjusted for age and advanced adenoma status (P < 0.01 for at least one marker component). When applied to the validation set, a predictive model using these seven markers showed substantial accuracy for identifying cases [area under the receiver operation characteristic curve (AUC), 0.83; 95% confidence interval (CI), 0.74-0.92]. A parsimonious model using three markers performed similarly to the seven-marker model (AUC, 0.84). In summary, we found that molecular markers of carcinogenesis distinguished adenomas from patients with and without colorectal cancer. Furthermore, we speculate that prospective studies using molecular markers to identify individuals with polyps at risk for future neoplasia are warranted. ©2014 American Association for Cancer Research.

The PI3K inhibitor GDC-0941 enhances radiosensitization and reduces chemoresistance to temozolomide in GBM cell lines.

PubMed

Shi, Fei; Guo, Hongchuan; Zhang, Rong; Liu, Hongyu; Wu, Liangliang; Wu, Qiyan; Liu, Jialin; Liu, Tianyi; Zhang, Qiuhang

2017-03-27

Glioblastoma multiforme (GBM) is among the most lethal of all human tumors. It is the most frequently occurring malignant primary brain tumor in adults. The current standard of care (SOC) for GBM is initial surgical resection followed by treatment with a combination of temozolomide (TMZ) and ionizing radiation (IR). However, GBM has a dismal prognosis, and survivors have compromised quality of life owing to the adverse effects of radiation. GBM is characterized by overt activity of the phosphoinositide 3-kinase (PI3K) signaling pathway. GDC-0941 is a highly specific PI3K inhibitor with promising anti-tumor activity in human solid tumors. It is being evaluated in Phase II clinical trials for the treatment of breast and non-squamous cell lung cancer. We hypothesized that GDC-0941 may act as an antitumor agent and potentiate the effects of TMZ and IR. In this study, GDC-0941 alone induced cytotoxicity and pro-apoptotic effects. Moreover, combined with the standard GBM therapy (TMZ and IR), it suppressed cell viability, showed enhanced pro-apoptotic effects, augmented autophagy response, and attenuated migratory/invasive capacity in three glioma cell lines. Protein microarray analyses showed that treatment with TMZ+GDC-0941+IR induced higher levels of p53 and glycogen synthase kinase 3-beta (GSK3-β) expression in SHG44GBM cells than those induced by other treatments. This was verified in all cell lines by western blot analysis. Furthermore, the combination of TMZ and GDC-0941 with or without IR reduced the levels of p-AKT and O 6 -methylguanine DNA methyltransferase (MGMT) in T98G cells. The results of this study suggest that the combination of TMZ, IR, and GDC-0941 is a promising choice for future treatments of GBM. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

Divergent evolution of temozolomide resistance in glioblastoma stem cells is reflected in extracellular vesicles and coupled with radiosensitization.

PubMed

Garnier, Delphine; Meehan, Brian; Kislinger, Thomas; Daniel, Paul; Sinha, Ankit; Abdulkarim, Bassam; Nakano, Ichiro; Rak, Janusz

2018-01-22

Glioblastoma (GBM) is almost invariably fatal due to failure of standard therapy. The relapse of GBM following surgery, radiation, and systemic temozolomide (TMZ) is attributed to the ability of glioma stem cells (GSCs) to survive, evolve, and repopulate the tumor mass, events on which therapy exerts a poorly understood influence. Here we explore the molecular and cellular evolution of TMZ resistance as it emerges in vivo (xenograft models) in a series of human GSCs with either proneural (PN) or mesenchymal (MES) molecular characteristics. We observed that the initial response of GSC-initiated intracranial xenografts to TMZ is eventually replaced by refractory growth pattern. Individual tumors derived from the same isogenic GSC line expressed divergent and complex profiles of TMZ resistance markers, with a minor representation of O6-methylguanine DNA methyltransferase (MGMT) upregulation. In several independent TMZ-resistant tumors originating from MES GSCs we observed a consistent diminution of mesenchymal features, which persisted in cell culture and correlated with increased expression of Nestin, decline in transglutaminase 2 and sensitivity to radiation. The corresponding mRNA expression profiles reflective of TMZ resistance and stem cell phenotype were recapitulated in the transcriptome of exosome-like extracellular vesicles (EVs) released by GSCs into the culture medium. Intrinsic changes in the tumor-initiating cell compartment may include loss of subtype characteristics and reciprocal alterations in sensitivity to chemo- and radiation therapy. These observations suggest that exploiting therapy-induced changes in the GSC phenotype and alternating cycles of therapy may be explored to improve GBM outcomes. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

Functional characterization of KanP, a methyltransferase from the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus.

PubMed

Nepal, Keshav Kumar; Yoo, Jin Cheol; Sohng, Jae Kyung

2010-09-20

KanP, a putative methyltransferase, is located in the kanamycin biosynthetic gene cluster of Streptomyces kanamyceticus ATCC12853. Amino acid sequence analysis of KanP revealed the presence of S-adenosyl-L-methionine binding motifs, which are present in other O-methyltransferases. The kanP gene was expressed in Escherichia coli BL21 (DE3) to generate the E. coli KANP recombinant strain. The conversion of external quercetin to methylated quercetin in the culture extract of E. coli KANP proved the function of kanP as S-adenosyl-L-methionine-dependent methyltransferase. This is the first report concerning the identification of an O-methyltransferase gene from the kanamycin gene cluster. The resistant activity assay and RT-PCR analysis demonstrated the leeway for obtaining methylated kanamycin derivatives from the wild-type strain of kanamycin producer. 2009 Elsevier GmbH. All rights reserved.

The protein arginine methyltransferase PRMT5 promotes D2-like dopamine receptor signaling

PubMed Central

Likhite, Neah; Jackson, Christopher A.; Liang, Mao-Shih; Krzyzanowski, Michelle C.; Lei, Pedro; Wood, Jordan F.; Birkaya, Barbara; Michaels, Kerry L.; Andreadis, Stelios T.; Clark, Stewart D.; Yu, Michael C.; Ferkey, Denise M.

2017-01-01

Protein arginine methylation regulates diverse functions of eukaryotic cells, including gene expression, the DNA damage response, and circadian rhythms. We showed that arginine residues within the third intracellular loop of the human D2 dopamine receptor, which are conserved in the DOP-3 receptor in the nematode Caenorhabditis elegans, were methylated by protein arginine methyl-transferase 5 (PRMT5). By mutating these arginine residues, we further showed that their methylation enhanced the D2 receptor–mediated inhibition of cyclic adenosine monophosphate (cAMP) signaling in cultured human embryonic kidney (HEK) 293T cells. Analysis of prmt-5–deficient worms indicated that methylation promoted the dopamine-mediated modulation of chemosensory and locomotory behaviors in C. elegans through the DOP-3 receptor. In addition to delineating a previously uncharacterized means of regulating GPCR (heterotrimeric guanine nucleotide–binding protein–coupled receptor) signaling, these findings may lead to the development of a new class of pharmacological therapies that modulate GPCR signaling by changing the methylation status of these key proteins. PMID:26554819

Combined radiotherapy and chemotherapy for high-grade brain tumours

NASA Astrophysics Data System (ADS)

Barazzuol, Lara

Glioblastoma (GBM) is the most common primary brain tumour in adults and among the most aggressive of all tumours. For several decades, the standard care of GBM was surgical resection followed by radiotherapy alone. In 2005, a landmark phase III clinical trial coordinated by the European Organization for Research and Treatment of Cancer (EORTC) and the National Cancer Institute of Canada (NCIC) demonstrated the benefit of radiotherapy with concomitant and adjuvant temozolomide (TMZ) chemotherapy. With TMZ, the median life expectancy in optimally managed patients is still only 12-14 months, with only 25% surviving 24 months. There is an urgent need for new therapies in particular in those patients whose tumour has an unmethylated methylguanine methyltransferase gene (MGMT) promoter, which is a predictive factor of benefit from TMZ. In this dissertation, the nature of the interaction between TMZ and radiation is investigated using both a mathematical model, based on in vivo population statistics of survival, and in vitro experimentation on a panel of human GBM cell lines. The results show that TMZ has an additive effect in vitro and that the population-based model may be insufficient in predicting TMZ response. The combination of TMZ with particle therapy is also investigated. Very little preclinical data exists on the effects of charged particles on GBM cell lines as well as on the concomitant application of chemotherapy. In this study, human GBM cells are exposed to 3 MeV protons and 6 MeV alpha particles in concomitance with TMZ. The results suggest that the radiation quality does not affect the nature of the interaction between TMZ and radiation, showing reproducible additive cytotoxicity. Since TMZ and radiation cause DNA damage in cancer cells, there has been increased attention to the use of poly(ADP-ribose) polymerase (PARP) inhibitors. PARP is a family of enzymes that play a key role in the repair of DNA breaks. In this study, a novel PARP inhibitor, ABT-888

The Ether-Cleaving Methyltransferase System of the Strict Anaerobe Acetobacterium dehalogenans: Analysis and Expression of the Encoding Genes▿

PubMed Central

Schilhabel, Anke; Studenik, Sandra; Vödisch, Martin; Kreher, Sandra; Schlott, Bernhard; Pierik, Antonio Y.; Diekert, Gabriele

2009-01-01

Anaerobic O-demethylases are inducible multicomponent enzymes which mediate the cleavage of the ether bond of phenyl methyl ethers and the transfer of the methyl group to tetrahydrofolate. The genes of all components (methyltransferases I and II, CP, and activating enzyme [AE]) of the vanillate- and veratrol-O-demethylases of Acetobacterium dehalogenans were sequenced and analyzed. In A. dehalogenans, the genes for methyltransferase I, CP, and methyltransferase II of both O-demethylases are clustered. The single-copy gene for AE is not included in the O-demethylase gene clusters. It was found that AE grouped with COG3894 proteins, the function of which was unknown so far. Genes encoding COG3894 proteins with 20 to 41% amino acid sequence identity with AE are present in numerous genomes of anaerobic microorganisms. Inspection of the domain structure and genetic context of these orthologs predicts that these are also reductive activases for corrinoid enzymes (RACEs), such as carbon monoxide dehydrogenase/acetyl coenzyme A synthases or anaerobic methyltransferases. The genes encoding the O-demethylase components were heterologously expressed with a C-terminal Strep-tag in Escherichia coli, and the recombinant proteins methyltransferase I, CP, and AE were characterized. Gel shift experiments showed that the AE comigrated with the CP. The formation of other protein complexes with the O-demethylase components was not observed under the conditions used. The results point to a strong interaction of the AE with the CP. This is the first report on the functional heterologous expression of acetogenic phenyl methyl ether-cleaving O-demethylases. PMID:19011025

Catechol-O-Methyltransferase Val158Met Polymorphism on Striatum Structural Covariance Networks in Alzheimer's Disease.

PubMed

Chang, Chiung-Chih; Tsai, Shih-Jen; Chen, Nai-Ching; Huang, Chi-Wei; Hsu, Shih-Wei; Chang, Ya-Ting; Liu, Mu-En; Chang, Wen-Neng; Tsai, Wan-Chen; Lee, Chen-Chang

2018-06-01

The catechol-O-methyltransferase enzyme metabolizes dopamine in the prefrontal axis, and its genetic polymorphism (rs4680; Val158Met) is a known determinant of dopamine signaling. In this study, we investigated the possible structural covariance networks that may be modulated by this functional polymorphism in patients with Alzheimer's disease. Structural covariance networks were constructed by 3D T1 magnetic resonance imaging. The patients were divided into two groups: Met-carriers (n = 91) and Val-homozygotes (n = 101). Seed-based analysis was performed focusing on triple-network models and six striatal networks. Neurobehavioral scores served as the major outcome factors. The role of seed or peak cluster volumes, or a covariance strength showing Met-carriers > Val-homozygotes were tested for the effect on dopamine. Clinically, the Met-carriers had higher mental manipulation and hallucination scores than the Val-homozygotes. The volume-score correlations suggested the significance of the putaminal seed in the Met-carriers and caudate seed in the Val-homozygotes. Only the dorsal-rostral and dorsal-caudal putamen interconnected peak clusters showed covariance strength interactions (Met-carriers > Val-homozygotes), and the peak clusters also correlated with the neurobehavioral scores. Although the triple-network model is important for a diagnosis of Alzheimer's disease, our results validated the role of the dorsal-putaminal-anchored network by the catechol-O-methyltransferase Val158Met polymorphism in predicting the severity of cognitive and behavior in subjects with Alzheimer's disease.

Properly Substituted Analogues of BIX-01294 Lose Inhibition of G9a Histone Methyltransferase and Gain Selective Anti-DNA Methyltransferase 3A Activity

PubMed Central

Rotili, Dante; Tarantino, Domenico; Marrocco, Biagina; Gros, Christina; Masson, Véronique; Poughon, Valérie; Ausseil, Fréderic; Chang, Yanqi; Labella, Donatella; Cosconati, Sandro; Di Maro, Salvatore; Novellino, Ettore; Schnekenburger, Michael; Grandjenette, Cindy; Bouvy, Celine; Diederich, Marc; Cheng, Xiaodong; Arimondo, Paola B.; Mai, Antonello

2014-01-01

Chemical manipulations performed on the histone H3 lysine 9 methyltransferases (G9a/GLP) inhibitor BIX-01294 afforded novel desmethoxyquinazolines able to inhibit the DNA methyltransferase DNMT3A at low micromolar levels without any significant inhibition of DNMT1 and G9a. In KG-1 cells such compounds, when tested at sub-toxic doses, induced the luciferase re-expression in a stable construct controlled by a cytomegalovirus (CMV) promoter silenced by methylation (CMV-luc assay). Finally, in human lymphoma U-937 and RAJI cells, the N-(1-benzylpiperidin-4-yl)-2-(4-phenylpiperazin-1-yl)quinazolin-4-amine induced the highest proliferation arrest and cell death induction starting from 10 µM, in agreement with its DNMT3A inhibitory potency. PMID:24810902

Boron neutron capture therapy for newly diagnosed glioblastoma multiforme: an assessment of clinical potential

PubMed Central

Sköld, K; Gorlia, T; Pellettieri, L; Giusti, V; H-Stenstam, B; Hopewell, J W

2010-01-01

The purpose of this study was to assess the potential of boron neutron capture therapy (BNCT), with a 6-h infusion of the boron carrier l-boronophenylalanine as a fructose preparation (BPA-f), as first-line radiotherapy for newly diagnosed glioblastoma multiforme (GBM). Patient survival data from a Phase II study using BNCT were compared with retrospective data from the two arms of a Phase III study using conventional radiotherapy (RT) in the reference arm and using RT plus concomitant and adjuvant medication with temozolomide (TMZ) in the experimental arm, and were also compared with small subgroups of these patients for whom the methylation status of the MGMT (O6-methylguanine–DNA methyltransferase) DNA repair gene was known. Differences in the baseline characteristics, salvage therapy after recurrence and levels of severe adverse events were also considered. The results indicate that BNCT offers a treatment that is at least as effective as conventional RT alone. For patients with an unmethylated MGMT DNA repair gene, a possible clinical advantage of BNCT over RT/TMZ was suggested. BNCT is a single-day treatment, which is of convenience to patients, with mild side effects, which would offer an initial 6 weeks of good-quality life during the time when patients would otherwise be undergoing daily treatments with RT and TMZ. It is suggested that the use of BNCT with a 6-h infusion of BPA-f should be explored in a stratified randomised Phase II trial in which patients with the unmethylated MGMT DNA repair gene are offered BNCT in the experimental arm and RT plus TMZ in the reference arm. PMID:20603410

Congenital thiopurine methyltransferase deficiency and 6-mercaptopurine toxicity during treatment for acute lymphoblastic leukaemia.

PubMed Central

Lennard, L; Gibson, B E; Nicole, T; Lilleyman, J S

1993-01-01

Two children with acute lymphoblastic leukaemia (ALL) taking daily 6-mercaptopurine as part of a national UK therapeutic trial repeatedly developed profound myelosuppression on 25% of the standard protocol dose. Both were found to have undetectable intracellular activity of thiopurine methyltransferase (TPMT), an enzyme controlling one of the major alternative catabolic pathways of 6-mercaptopurine, and both produced higher concentrations of cytotoxic drug metabolites at 10-25% of the protocol dose than other patients taking 100%. It is supposed that these patients represent the 0.33% of the normal population constitutionally lacking TPMT. It is important to recognise such individuals both to avoid fatal bone marrow failure through inadvertent overdosage, and to be reassured that an adequate drug effect can be achieved at around 10% of the standard dose. PMID:8257179

Double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours.

PubMed

Burke, Elinor; Grobler, Mariana; Elderfield, Kay; Bond, Frances; Crocker, Matthew; Taylor, Rohan; Bridges, Leslie R

2013-06-10

Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard.

Ribosomal Proteins RPS11 and RPS20, Two Stress-Response Markers of Glioblastoma Stem Cells, Are Novel Predictors of Poor Prognosis in Glioblastoma Patients

PubMed Central

Yang, Shuai; Tso, Jonathan L.; Menjivar, Jimmy C.; Wei, Bowen; Lucey, Gregory M.; Mareninov, Sergey; Chen, Zugen; Liau, Linda M.; Lai, Albert; Nelson, Stanley F.; Cloughesy, Timothy F.; Tso, Cho-Lea

2015-01-01

Glioblastoma stem cells (GSC) co-exhibiting a tumor-initiating capacity and a radio-chemoresistant phenotype, are a compelling cell model for explaining tumor recurrence. We have previously characterized patient-derived, treatment-resistant GSC clones (TRGC) that survived radiochemotherapy. Compared to glucose-dependent, treatment-sensitive GSC clones (TSGC), TRGC exhibited reduced glucose dependence that favor the fatty acid oxidation pathway as their energy source. Using comparative genome-wide transcriptome analysis, a series of defense signatures associated with TRGC survival were identified and verified by siRNA-based gene knockdown experiments that led to loss of cell integrity. In this study, we investigate the prognostic value of defense signatures in glioblastoma (GBM) patients using gene expression analysis with Probeset Analyzer (131 GBM) and The Cancer Genome Atlas (TCGA) data, and protein expression with a tissue microarray (50 GBM), yielding the first TRGC-derived prognostic biomarkers for GBM patients. Ribosomal protein S11 (RPS11), RPS20, individually and together, consistently predicted poor survival of newly diagnosed primary GBM tumors when overexpressed at the RNA or protein level [RPS11: Hazard Ratio (HR) = 11.5, p<0.001; RPS20: HR = 4.5, p = 0.03; RPS11+RPS20: HR = 17.99, p = 0.001]. The prognostic significance of RPS11 and RPS20 was further supported by whole tissue section RPS11 immunostaining (27 GBM; HR = 4.05, p = 0.01) and TCGA gene expression data (578 primary GBM; RPS11: HR = 1.19, p = 0.06; RPS20: HR = 1.25, p = 0.02; RPS11+RPS20: HR = 1.43, p = 0.01). Moreover, tumors that exhibited unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) or wild-type isocitrate dehydrogenase 1 (IDH1) were associated with higher RPS11 expression levels [corr (IDH1, RPS11) = 0.64, p = 0.03); [corr (MGMT, RPS11) = 0.52, p = 0.04]. These data indicate that increased expression of RPS11 and RPS20 predicts shorter patient survival. The study also

Association between TPMT*3C and decreased thiopurine S-methyltransferase activity in patients with neuromyelitis optica spectrum disorders in China.

PubMed

Gong, Xiaoqing; Mei, Shenghui; Li, Xindi; Li, Xingang; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Yang, Li; Zhao, Zhigang; Zhang, Xinghu

2018-06-01

Thiopurines are effective drugs in treating neuromyelitis optica spectrum disorders and other diseases. Thiopurines' toxicity is mainly imputed to thiopurine S-methyltransferase activity. In Chinese population, the most common and important variation of thiopurine S-methyltransferase is TPMT*3C (rs1142345). This study aims to reveal the association between thiopurine S-methyltransferase activity and genetic polymorphisms of thiopurine S-methyltransferase in patients with neuromyelitis optica spectrum disorders in China. A liquid chromatography tandem mass/mass method was used to evaluate the thiopurine S-methyltransferase activity by using 6-mercapthioprine as the substrate in human erythrocyte haemolysate via 1 h incubation at 37 °C to form its methylated product 6-methylmercaptopurine. The amount of 6-methylmercaptopurine was adjusted by haematocrit and normalized to 8 × 10 8 erythrocytes. The selected polymorphisms of thiopurine S-methyltransferase were identified using MassARRAY system (Sequenom) and multiple SNaPshot technique. In 69 patients with neuromyelitis optica spectrum disorders, thiopurine S-methyltransferase activity was 80.29-154.53 (127.51 ± 16.83) pmol/h/8 × 10 8 erythrocytes. TPMT*3C (rs1142345) was associated with lower thiopurine S-methyltransferase activity (BETA = -25.37, P = 0.011). Other selected variants were not associated with thiopurine S-methyltransferase activity. TPMT*3C affects TPMT activity in Chinese patients with neuromyelitis optica spectrum disorders. Further studies are warranted to confirm the results. TPRs = thiopurines; NMOSD = neuromyelitis optica spectrum disorders; TPMT = thiopurine S-methyltransferase; LC-MS/MS = liquid chromatography tandem mass/mass; 6-MMP = 6-methylmercaptopurine; IS = internal standard; SNP = single nucleotide polymorphism; MAF = minor allele frequency; HWE = Hardy-Weinberg equilibrium; BETA = regression coefficients; UTR-3 = untranslated region 3.

Characterization of regioselective flavonoid O-methyltransferase from the Streptomyces sp. KCTC 0041BP.

PubMed

Darsandhari, Sumangala; Dhakal, Dipesh; Shrestha, Biplav; Parajuli, Prakash; Seo, Joo-Hyun; Kim, Tae-Su; Sohng, Jae Kyung

2018-06-01

A flavonoid comprises polyphenol compounds with pronounced antiviral, antioxidant, anticarcinogenic, and anti-inflammatory effects. The flavonoid modification by methylation provides a greater stability and improved pharmacokinetic properties. The methyltransferase from plants or microorganisms is responsible for such substrate modifications in a regiospecific or a promiscuous manner. GerMIII, originally characterized as a putative methyltransferase in a dihydrochalcomycin biosynthetic gene cluster of the Streptomyces sp. KCTC 0041BP, was tested for the methylation of the substrates of diverse chemical structures. Among the various tested substrates, flavonoids emerged as the favored substrates for methylation. Further, among the flavonoids, quercetin is the most favorable substrate, followed by luteolin, myricetin, quercetin 3-O-β-D-glucoside, and fisetin, while only a single product was formed in each case. The products were confirmed by HPLC and mass-spectrometry analyses. A detailed NMR spectrometric analysis of the methylated quercetin and luteolin derivatives confirmed the regiospecific methylation at the 4'-OH position. Modeling and molecular docking provided further insight regarding the most favorable mechanism and substrate architecture for the enzymatic catalysis. Accordingly, a double bond between the C 2 and the C 3 and a single-ring-appended conjugate-hydroxyl group are crucial for the favorable enzymatic conversions of the GerMIII catalysis. Thus, in this study, the enzymatic properties of GerMIII and a mechanistic overview of the regiospecific modification that was implemented for the acceptance of quercetin as the most favorable substrate are presented. Copyright © 2018 Elsevier Inc. All rights reserved.

No association between catechol-O-methyltransferase polymorphisms and neurotic disorders among mainland Chinese university students.

PubMed

Kou, Changgui; Meng, Xiangfei; Xie, Bing; Shi, Jieping; Yu, Qiong; Yu, Yaqin; D'Arcy, Carl

2012-07-30

This study investigates the genetic association between catechol-O-methyltransferase (COMT) gene polymorphisms and neurotic disorders. Data were derived from a case-control association study of 255 undergraduates affected by neurotic disorders and 269 matched healthy undergraduate controls. The polymorphisms of eight tag single nucleotide polymorphisms (SNPs) on the COMT gene were tested using polymerase chain reaction (PCR)-based Ligase Detection Reaction (PCR-LDR). The eight tag SNPs on the COMT gene assessed were not associated with neurotic disorders. Our finding suggests that the COMT gene may not be a susceptibility gene for neurotic disorders. Copyright © 2012 Elsevier Ltd. All rights reserved.

Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus.

PubMed Central

Kanugula, Sreenivas; Pegg, Anthony E

2003-01-01

AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy. PMID:12892560

Differential DNA methylation of the meiosis-specific gene FKBP6 in testes of yak and cattle-yak hybrids.

PubMed

Li, B; Luo, H; Weng, Q; Wang, S; Pan, Z; Xie, Z; Wu, W; Liu, H; Li, Q

2016-12-01

FK506-binding protein 6 (FKBP6) is essential for meiosis during mammalian spermatogenesis. However, the molecular regulation of FKBP6 during spermatogenesis remains unclear. In the present study, we performed molecular characterization of the meiosis-specific gene FKBP6 in yak testes. Yak FKBP6 encodes a polypeptide of 295 amino acid residues with an FK506-binding domain (FKBP_C) and three tetratricopeptide repeat domains. The methylation level of the FKBP6 promoter in testes was significantly higher in cattle-yak with male sterility than in yak, and the FKBP6 promoter was methylated in liver tissues in which FKBP6 is not expressed. FKBP6 promoter activity was significantly decreased after treatment with the M.SssI methyltransferase in vitro. Furthermore, the FKBP6 gene was remarkably activated in bovine mammary epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2-deoxycytidine. Taken together, our results demonstrate for the first time that the FKBP6 promoter is differentially methylated in testes; together with the functional promoter analysis, this suggests that methylation of this promoter may contribute to cattle-yak male infertility. © 2016 Blackwell Verlag GmbH.

Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

PubMed Central

2013-01-01

Background Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). Results Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. Conclusions Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard. PMID:24252243

Inducible repair of alkylated DNA in microorganisms.

PubMed

Mielecki, Damian; Wrzesiński, Michał; Grzesiuk, Elżbieta

2015-01-01

Alkylating agents, which are widespread in the environment, also occur endogenously as primary and secondary metabolites. Such compounds have intrinsically extremely cytotoxic and frequently mutagenic effects, to which organisms have developed resistance by evolving multiple repair mechanisms to protect cellular DNA. One such defense against alkylation lesions is an inducible Adaptive (Ada) response. In Escherichia coli, the Ada response enhances cell resistance by the biosynthesis of four proteins: Ada, AlkA, AlkB, and AidB. The glycosidic bonds of the most cytotoxic lesion, N3-methyladenine (3meA), together with N3-methylguanine (3meG), O(2)-methylthymine (O(2)-meT), and O(2)-methylcytosine (O(2)-meC), are cleaved by AlkA DNA glycosylase. Lesions such as N1-methyladenine (1meA) and N3-methylcytosine (3meC) are removed from DNA and RNA by AlkB dioxygenase. Cytotoxic and mutagenic O(6)-methylguanine (O(6)meG) is repaired by Ada DNA methyltransferase, which transfers the methyl group onto its own cysteine residue from the methylated oxygen. We review (i) the individual Ada proteins Ada, AlkA, AlkB, AidB, and COG3826, with emphasis on the ubiquitous and versatile AlkB and its prokaryotic and eukaryotic homologs; (ii) the organization of the Ada regulon in several bacterial species; (iii) the mechanisms underlying activation of Ada transcription. In vivo and in silico analysis of various microorganisms shows the widespread existence and versatile organization of Ada regulon genes, including not only ada, alkA, alkB, and aidB but also COG3826, alkD, and other genes whose roles in repair of alkylated DNA remain to be elucidated. This review explores the comparative organization of Ada response and protein functions among bacterial species beyond the classical E. coli model. Copyright © 2014 Elsevier B.V. All rights reserved.

Genetic Manipulation of Isoflavone 7-O-Methyltransferase Enhances Biosynthesis of 4′-O-Methylated Isoflavonoid Phytoalexins and Disease Resistance in Alfalfa

PubMed Central

He, Xian-Zhi; Dixon, Richard A.

2000-01-01

4′-O-Methylation of an isoflavonoid intermediate is a key reaction in the biosynthesis of the phytoalexin medicarpin in legumes. However, isoflavone O-methyltransferase (IOMT) from alfalfa converts the isoflavone daidzein to 7-O-methyl daidzein (isoformononetin) in vitro as well as in vivo in unchallenged leaves of transgenic alfalfa ectopically expressing IOMT. In contrast, elicitation of IOMT-overexpressing plants with CuCl2 or infecting these plants with Phoma medicaginis leads to greater accumulation of formononetin (4′-O-methyl daidzein) and medicarpin in the leaves than does elicitation or infection of control plants, and no isoformononetin is detected. Overexpression of IOMT results in increased induction of phenylpropanoid/isoflavonoid pathway gene transcripts after infection but has little effect on basal expression of these genes. IOMT-overexpressing plants display resistance to P. medicaginis. The apparently different regiospecificities of IOMT in vivo and in vitro are discussed in relation to potential metabolic channeling at the entry point into the isoflavonoid pathway. PMID:11006341

Computational Investigation of the Interplay of Substrate Positioning and Reactivity in Catechol O-Methyltransferase

PubMed Central

Patra, Niladri; Ioannidis, Efthymios I.

2016-01-01

Catechol O-methyltransferase (COMT) is a SAM- and Mg2+-dependent methyltransferase that regulates neurotransmitters through methylation. Simulations and experiments have identified divergent catecholamine substrate orientations in the COMT active site: molecular dynamics simulations have favored a monodentate coordination of catecholate substrates to the active site Mg2+, and crystal structures instead preserve bidentate coordination along with short (2.65 Å) methyl donor-acceptor distances. We carry out longer dynamics (up to 350 ns) to quantify interconversion between bidentate and monodentate binding poses. We provide a systematic determination of the relative free energy of the monodentate and bidentate structures in order to identify whether structural differences alter the nature of the methyl transfer mechanism and source of enzymatic rate enhancement. We demonstrate that the bidentate and monodentate binding modes are close in energy but separated by a 7 kcal/mol free energy barrier. Analysis of interactions in the two binding modes reveals that the driving force for monodentate catecholate orientations in classical molecular dynamics simulations is derived from stronger electrostatic stabilization afforded by alternate Mg2+ coordination with strongly charged active site carboxylates. Mixed semi-empirical-classical (SQM/MM) substrate C-O distances (2.7 Å) for the bidentate case are in excellent agreement with COMT X-ray crystal structures, as long as charge transfer between the substrates, Mg2+, and surrounding ligands is permitted. SQM/MM free energy barriers for methyl transfer from bidentate and monodentate catecholate configurations are comparable at around 21–22 kcal/mol, in good agreement with experiment (18–19 kcal/mol). Overall, the work suggests that both binding poses are viable for methyl transfer, and accurate descriptions of charge transfer and electrostatics are needed to provide balanced relative barriers when multiple binding poses are

Substrate Scope of O-Methyltransferase from Streptomyces peucetius for Biosynthesis of Diverse Natural Products Methoxides.

PubMed

Parajuli, Prakash; Pandey, Ramesh Prasad; Nguyen, Thi Huyen Trang; Dhakal, Dipesh; Sohng, Jae Kyung

2018-04-01

Methylation is a common post-modification reaction that is observed during the biosynthesis of secondary metabolites produced by plants and microorganisms. Based on the sequence information from Streptomyces peucetius ATCC27952, a putative O-methyltransferase (OMT) gene SpOMT7740 was polymerase chain reaction amplified and cloned into E. coli BL21 (DE3) host to test the substrate promiscuity and conduct functional characterization. In vitro and in vivo reaction assays were carried out over various classes of substrates: flavonoids (flavonol, flavones, and isoflavonoid), chalcones, anthraquinones, anthracyclines, and sterol molecules, and the applications in synthesizing diverse classes of O-methoxy natural products were also illustrated. SpOMT7740 catalyzed the O-methylation reaction to form various natural and non-natural O-methoxides, includes 7-hydroxy-8-O-methoxy flavone, 3-O-methoxy flavone, three mono-, di-, and tri-O-methoxy genistein, mono-O-methoxy phloretin, mono-O-methoxy luteolin, 3-O-methoxy β-sitosterol, and O-methoxy anthraquinones (emodin and aloe emodin) and O-methoxy anthracycline (daunorubicin) exhibiting diverse substrate flexibility. Daunorubicin is a native secondary metabolite of S. peucetius. Among the compounds tested, 7,8-dihydroxyflavone was the best substrate for bioconversion to 7-hydroxy-8-O-methoxy flavone, and it was structurally elucidated. This enzyme showed a flexible catalysis over the given ranges of temperature, pH, and divalent cationic conditions for O-methylation.

RNA Cap Methyltransferase Activity Assay

PubMed Central

Trotman, Jackson B.; Schoenberg, Daniel R.

2018-01-01

Methyltransferases that methylate the guanine-N7 position of the mRNA 5′ cap structure are ubiquitous among eukaryotes and commonly encoded by viruses. Here we provide a detailed protocol for the biochemical analysis of RNA cap methyltransferase activity of biological samples. This assay involves incubation of cap-methyltransferase-containing samples with a [32P]G-capped RNA substrate and S-adenosylmethionine (SAM) to produce RNAs with N7-methylated caps. The extent of cap methylation is then determined by P1 nuclease digestion, thin-layer chromatography (TLC), and phosphorimaging. The protocol described here includes additional steps for generating the [32P]G-capped RNA substrate and for preparing nuclear and cytoplasmic extracts from mammalian cells. This assay is also applicable to analyzing the cap methyltransferase activity of other biological samples, including recombinant protein preparations and fractions from analytical separations and immunoprecipitation/pulldown experiments. PMID:29644259

Expression of DNA repair proteins MSH2, MLH1 and MGMT in human benign and malignant thyroid lesions: An immunohistochemical study

PubMed Central

Giaginis, Constantinos; Michailidi, Christina; Stolakis, Vasileios; Alexandrou, Paraskevi; Tsourouflis, Gerasimos; Klijanienko, Jerzy; Delladetsima, Ioanna; Theocharis, Stamatios

2011-01-01

Summary Background DNA repair is a major defense mechanism, which contributes to the maintenance of genetic sequence, and minimizes cell death, mutation rates, replication errors, DNA damage persistence and genomic instability. Alterations in the expression levels of proteins participating in DNA repair mechanisms have been associated with several aspects of cancer biology. The present study aimed to evaluate the clinical significance of DNA repair proteins MSH2, MLH1 and MGMT in benign and malignant thyroid lesions. Material/Methods MSH2, MLH1 and MGMT protein expression was assessed immunohistochemically on paraffin-embedded thyroid tissues from 90 patients with benign and malignant lesions. Results The expression levels of MLH1 was significantly upregulated in cases with malignant compared to those with benign thyroid lesions (p=0.038). The expression levels of MGMT was significantly downregulated in malignant compared to benign thyroid lesions (p=0.001). Similar associations for both MLH1 and MGMT between cases with papillary carcinoma and hyperplastic nodules were also noted (p=0.014 and p=0.026, respectively). In the subgroup of malignant thyroid lesions, MSH2 downregulation was significantly associated with larger tumor size (p=0.031), while MLH1 upregulation was significantly associated with the presence of lymphatic and vascular invasion (p=0.006 and p=0.002, respectively). Conclusions Alterations in the mismatch repair proteins MSH2 and MLH1 and the direct repair protein MGMT may result from tumor development and/or progression. Further studies are recommended to draw definite conclusions on the clinical significance of DNA repair proteins in thyroid neoplasia. PMID:21358597

Dopamine receptor D2 (DRD2), dopamine transporter solute carrier family C6, member 4 (SLC6A3), and catechol-O-methyltransferase (COMT) genes as moderators of the relation between maternal history of maltreatment and infant emotion regulation.

PubMed

Villani, Vanessa; Ludmer, Jaclyn; Gonzalez, Andrea; Levitan, Robert; Kennedy, James; Masellis, Mario; Basile, Vincenzo S; Wekerle, Christine; Atkinson, Leslie

2018-05-01

Although infants less than 18 months old are capable of engaging in self-regulatory behavior (e.g., avoidance, withdrawal, and orienting to other aspects of their environment), the use of self-regulatory strategies at this age (as opposed to relying on caregivers) is associated with elevated behavioral and physiological distress. This study investigated infant dopamine-related genotypes (dopamine receptor D2 [DRD2], dopamine transporter solute carrier family C6, member 4 [SLC6A3], and catechol-O-methyltransferase [COMT]) as they interact with maternal self-reported history of maltreatment to predict observed infant independent emotion regulation behavior. A community sample (N = 193) of mother-infant dyads participated in a toy frustration challenge at infant age 15 months, and infant emotion regulation behavior was coded. Buccal cells were collected for genotyping. Maternal maltreatment history significantly interacted with infant SLC6A3 and COMT genotypes, such that infants with more 10-repeat and valine alleles of SLC6A3 and COMT, respectively, relative to infants with fewer or no 10-repeat and valine alleles, utilized more independent (i.e., maladaptive) regulatory behavior if mother reported a more extensive maltreatment history, as opposed to less. The findings indicate that child genetic factors moderate the intergenerational impact of maternal maltreatment history. The results are discussed in terms of potential mechanism of Gene × Environment interaction.

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

PubMed

Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

2009-03-04

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

Functional and Structural Characterization of a Cation-dependent O-Methyltransferase from the Cyanobacterium Synechocystis sp. Strain PCC 6803*S⃞

PubMed Central

Kopycki, Jakub Grzegorz; Stubbs, Milton T.; Brandt, Wolfgang; Hagemann, Martin; Porzel, Andrea; Schmidt, Jürgen; Schliemann, Willibald; Zenk, Meinhart H.; Vogt, Thomas

2008-01-01

The coding sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 slr0095 gene was cloned and functionally expressed in Escherichia coli. The corresponding enzyme was classified as a cation- and S-adenosyl-l-methionine-dependent O-methyltransferase (SynOMT), consistent with considerable amino acid sequence identities to eukaryotic O-methyltransferases (OMTs). The substrate specificity of SynOMT was similar with those of plant and mammalian CCoAOMT-like proteins accepting a variety of hydroxycinnamic acids and flavonoids as substrates. In contrast to the known mammalian and plant enzymes, which exclusively methylate the meta-hydroxyl position of aromatic di- and trihydroxy systems, Syn-OMT also methylates the para-position of hydroxycinnamic acids like 5-hydroxyferulic and 3,4,5-trihydroxycinnamic acid, resulting in the formation of novel compounds. The x-ray structure of SynOMT indicates that the active site allows for two alternative orientations of the hydroxylated substrates in comparison to the active sites of animal and plant enzymes, consistent with the observed preferred para-methylation and position promiscuity. Lys3 close to the N terminus of the recombinant protein appears to play a key role in the activity of the enzyme. The possible implications of these results with respect to modifications of precursors of polymers like lignin are discussed. PMID:18502765

Protein arginine methyltransferase 6 specifically methylates the nonhistone chromatin protein HMGA1a.

PubMed

Miranda, Tina Branscombe; Webb, Kristofor J; Edberg, Dale D; Reeves, Raymond; Clarke, Steven

2005-10-28

The HMGA family proteins HMGA1a and HMGA1b are nuclear nonhistone species implicated in a wide range of cellular processes including inducible gene transcription, modulation of chromosome structure through nucleosome and chromosome remodeling, and neoplastic transformation. HMGA proteins are highly modified, and changes in their phosphorylation states have been correlated with the phase of the cell cycle and changes in their transcriptional activity. HMGA1a is also methylated in the first DNA-binding AT-hook at Arg25 and other sites, although the enzyme or enzymes responsible have not been identified. We demonstrate here that a GST fusion of protein arginine methyltransferase 6 (PRMT6) specifically methylates full-length recombinant HMGA1a protein in vitro. Although GST fusions of PRMT1 and PRMT3 were also capable of methylating the full-length HMGA1a polypeptide, they recognize its proteolytic degradation products much better. GST fusions of PRMT4 or PRMT7 were unable to methylate the full-length protein or its degradation products. We conclude that PRMT6 is a good candidate for the endogenous enzyme responsible for HGMA1a methylation.

Determination of the structure and catalytic mechanism of Sorghum bicolor caffeic acid O-methyltransferase and the structural impact of three brown midrib12 mutations

USDA-ARS?s Scientific Manuscript database

With S-adenosylmethionine (SAM) acting as the methyl donor, caffeic acid O-methyltransferase from Sorghum bicolor (SbCOMT) methylates the 5-hydroxyl group of its preferred substrate, 5-hydroxyconiferaldehyde, to form sinapaldehyde. In order to determine the mechanism of SbCOMT and understand the red...

Methylation of 6-mercaptopurine and 6-thioguanine by thiopurine S-methyltransferase. A comparison of activity in red blood cell samples of 199 blood donors.

PubMed

Kröplin, T; Iven, H

2000-07-01

To compare 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) as substrates for the methylation reaction catalysed by the enzyme thiopurine S-methyltransferase (TPMT). TPMT activity in haemolysed red blood cells of healthy blood donors was determined twice, using the same experimental setting and equal molar concentrations of 6-TG and 6-MP as substrates. After extraction, the reaction products 6-methyl-TG and 6-methyl-MP were quantified using specific high-performance liquid chromatography procedures. The medians of the TPMT activities from 199 blood donors were 54.4 nmol 6-MTG g(-1)Hb h(-1) when measured with 6-TG as the substrate and 35.8 nmol 6-MMP g(-1) Hb h(-1) when measured with 6-MP. The correlation coefficient for the 199 pairs of values was 0.8695. On average, TPMT activity was 34% lower with 6-MP as substrate than with 6-TG as substrate.

Chloroplast overexpression of rice caffeic acid O-methyltransferase increases melatonin production in chloroplasts via the 5-methoxytryptamine pathway in transgenic rice plants.

PubMed

Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

2017-08-01

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5-MT) pathway. 5-MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase (SNAT). Here, we show that 5-MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5-MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5-MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5-MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5-MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mesenchymal stem cells cancel azoxymethane-induced tumor initiation.

PubMed

Nasuno, Masanao; Arimura, Yoshiaki; Nagaishi, Kanna; Isshiki, Hiroyuki; Onodera, Kei; Nakagaki, Suguru; Watanabe, Shuhei; Idogawa, Masashi; Yamashita, Kentaro; Naishiro, Yasuyoshi; Adachi, Yasushi; Suzuki, Hiromu; Fujimiya, Mineko; Imai, Kohzoh; Shinomura, Yasuhisa

2014-04-01

The role of mesenchymal stem cells (MSCs) in tumorigenesis remains controversial. Therefore, our goal was to determine whether exogenous MSCs possess intrinsic antineoplastic or proneoplastic properties in azoxymethane (AOM)-induced carcinogenesis. Three in vivo models were studied: an AOM/dextran sulfate sodium colitis-associated carcinoma model, an aberrant crypt foci model, and a model to assess the acute apoptotic response of a genotoxic carcinogen (AARGC). We also performed in vitro coculture experiments. As a result, we found that MSCs partially canceled AOM-induced tumor initiation but not tumor promotion. Moreover, MSCs inhibited the AARGC in colonic epithelial cells because of the removal of O(6)-methylguanine (O(6) MeG) adducts through O(6) MeG-DNA methyltransferase activation. Furthermore, MSCs broadly affected the cell-cycle machinery, potentially leading to G1 arrest in vivo. Coculture of IEC-6 rat intestinal cells with MSCs not only arrested the cell cycle at the G1 phase, but also induced apoptosis. The anti-carcinogenetic properties of MSCs in vitro required transforming growth factor (TGF)-β signaling because such properties were completely abrogated by absorption of TGF-β under indirect coculture conditions. MSCs inhibited AOM-induced tumor initiation by preventing the initiating cells from sustaining DNA insults and subsequently inducing G1 arrest in the initiated cells that escaped from the AARGC. Furthermore, tumor initiation perturbed by MSCs might potentially dysregulate WNT and TGF-β-Smad signaling pathways in subsequent tumorigenesis. Obtaining a better understanding of MSC functions in colon carcinogenesis is essential before commencing the broader clinical application of promising MSC-based therapies for cancer-prone patients with inflammatory bowel disease. © AlphaMed Press.

Dietary butyrylated high-amylose starch reduces azoxymethane-induced colonic O(6)-methylguanine adducts in rats as measured by immunohistochemistry and high-pressure liquid chromatography.

PubMed

Le Leu, Richard K; Scherer, Benjamin L; Mano, Mark T; Winter, Jean M; Lannagan, Tamsin; Head, Richard J; Lockett, Trevor; Clarke, Julie M

2016-09-01

O(6)-methyl guanine (O(6)MeG) adducts are major toxic, promutagenic, and procarcinogenic adducts involved in colorectal carcinogenesis. Resistant starch and its colonic metabolite butyrate are known to protect against oncogenesis in the colon. In this study, we hypothesized that a dietary intervention that specifically delivers butyrate to the large bowel (notably butyrylated high-amylose maize starch [HAMSB]) would reduce colonic levels of O(6)MeG in rats shortly after exposure to the deoxyribonucleic acid (DNA) alkylating agent azoxymethane (AOM) when compared with a low-amylose maize starch (LAMS). A further objective was to validate an immunohistochemistry (IHC) method for quantifying O(6)MeG against a high-performance liquid chromatography method using fluorescence and diode array detection. Rats were fed either LAMS or HAMSB diets for 4 weeks followed by a single injection of AOM or saline and killed 6 hours later. After AOM exposure, both IHC and high-performance liquid chromatography method using fluorescence and diode array detection measured a substantially increased quantity of DNA adducts in the colon (P<.001). Both techniques demonstrated equally that consumption of HAMSB provided a protective effect by reducing colonic adduct load compared with the LAMS diet (P<.05). In addition, IHC allowed visualization of the O(6)MeG distribution, where adduct load was reduced in the lower third of the crypt compartment in HAMSB-fed rats (P=.036). The apoptotic response to AOM was higher in the HAMSB-fed rats (P=.002). In conclusion, the reduction in O(6)MeG levels and enhancement of the apoptotic response to DNA damage in the colonic epithelium through consumption of HAMSB provide mechanistic insights into how HAMSB protects against colorectal tumorigenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis.

PubMed

Kobayashi, Masatoshi; Ohsugi, Mitsuru; Sasako, Takayoshi; Awazawa, Motoharu; Umehara, Toshihiro; Iwane, Aya; Kobayashi, Naoki; Okazaki, Yukiko; Kubota, Naoto; Suzuki, Ryo; Waki, Hironori; Horiuchi, Keiko; Hamakubo, Takao; Kodama, Tatsuhiko; Aoe, Seiichiro; Tobe, Kazuyuki; Kadowaki, Takashi; Ueki, Kohjiro

2018-06-04

Adipocyte differentiation is regulated by various mechanisms, of which the mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by the cell cycle modulators, the precise mechanism remains unclear. N 6 -methyladenosine (m 6 A) post-transcriptional RNA modification, whose methylation and demethylation is performed by respective enzymal molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N 6 -adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like (METTL) 3 and METTL14 positively control adipogenesis, by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of Cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistently, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders. Copyright © 2018 Kobayashi et al.

Catechol-O-methyltransferase (COMT) gene modulates private self-consciousness and self-flexibility.

PubMed

Wang, Bei; Ru, Wenzhao; Yang, Xing; Yang, Lu; Fang, Pengpeng; Zhu, Xu; Shen, Guomin; Gao, Xiaocai; Gong, Pingyuan

2016-08-01

Dopamine levels in the brain influence human consciousness. Inspired by the role of Catechol-O-methyltransferase (COMT) in inactivating dopamine in the brain, we investigated to what extent COMT could modulate individual's self-consciousness dispositions and self-consistency by genotyping the COMT Val158Met (rs4680) polymorphism and measuring self-consciousness and self-consistency and congruence in a college student population. The results indicated that COMT Val158Met polymorphism significantly modulated the private self-consciousness. The individuals with Val/Val genotype, corresponding to lower dopamine levels in the brain, were more likely to be aware of their feelings and beliefs. The results also indicated that this polymorphism modulated one's self-flexibility. The individuals with Val/Val genotype showed higher levels of stereotype in self-concept compared with those with Met/Met genotype. These findings suggest that COMT is a predictor of the individual differences in self-consciousness and self-flexibility. Copyright © 2016 Elsevier Inc. All rights reserved.

Functional characterization of O-methyltransferases used to catalyse site-specific methylation in the post-tailoring steps of pradimicin biosynthesis.

PubMed

Han, J W; Ng, B G; Sohng, J K; Yoon, Y J; Choi, G J; Kim, B S

2018-01-01

To identify the roles of the two O-methyltransferase homologous genes pdmF and pdmT in the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2. Pradimicins are pentangular polyphenol antibiotics synthesized by bacterial type II polyketide synthases (PKSs) and tailoring enzymes. Pradimicins are naturally derivatized by combinatorial O-methylation at two positions (i.e., 7-OH and 11-OH) of the benzo[α]naphthacenequinone structure. PdmF and PdmT null mutants (PFKO and PTKO) were generated. PFKO produced the 11-O-demethyl shunt metabolites 11-O-demethylpradimicinone II (1), 11-O-demethyl-7-methoxypradimicinone II (2), 11-O-demethylpradimicinone I (3) and 11-O-demethylpradimicin A (4), while PTKO generated the 7-O-demethyl derivatives pradimicinone II (5) and 7-hydroxypradimicin A (6). Pradimicinones 1, 2, 3, and 5 were fed to a heterologous host Escherichia coli harbouring expression plasmid pET-22b::pdmF or pET-28a::pdmT. PdmF catalysed 11-O-methylation of pradimicinones 1, 2, and 3 regardless of O-methylation at the C-7 position, while PdmT was unable to catalyse 7-O-methylation when the C-11 hydroxyl group was methylated (5). PdmF and PdmT were involved in 11-O- and 7-O-methylations of the benzo[α]naphthacenequinone moiety of pradimicin, respectively. Methylation of the C-7 hydroxyl group precedes methylation of the C-11 hydroxyl group in pradimicin biosynthesis. This is the first reported demonstration of the functions of PdmF and PdmT for regiospecific O-methylation, which contributes to better understanding of the post-PKS modifications in pradimicin biosynthesis as well as to rational engineering of the pradimicin biosynthetic machinery. © 2017 The Society for Applied Microbiology.

DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor.

PubMed

Matsuoka, Taeko; Kawai, Koji; Ando, Satoshi; Sugita, Shintaro; Kandori, Shuya; Kojima, Takahiro; Miyazaki, Jun; Nishiyama, Hiroyuki

2016-05-01

DNA methyltransferase 3-like plays an important role in germ cell development. The aim of this study was to analyse the DNA methyltransferase 3-like protein expression in testicular germ cell tumors. The immunohistochemical expression of DNA methyltransferase 3-like was examined in 86 testicular germ cell tumor specimens in various clinical settings. The association between DNA methyltransferase 3-like expression and disease stage was analyzed. DNA methyltransferase 3-like was strongly expressed in seven of the eight pure embryonal carcinomas (87.5%). Partial DNA methyltransferase 3-like expression was observed in 6 of 23 (26.1%) pure seminomas. Various degrees of DNA methyltransferase 3-like expression was observed in all four pure yolk sac tumors, of which three were prepubertal yolk sac tumors. In mixed germ cell tumors, DNA methyltransferase 3-like protein was expressed in various degrees in elements of the embryonal carcinoma (14/18, 77.8%), seminoma (4/11, 36.4%), teratoma (4/7, 57.1%) and choriocarcinoma (3/3, 100%) but not in the yolk sac tumors (0/4). When DNA methyltransferase 3-like expression was analyzed according to disease stages, it was significantly correlated with advanced seminoma rather than Stage I seminoma (46.2 vs. 0%, P = 0.019), whereas there was no significant difference in the DNA methyltransferase 3-like-positive proportion between Stage I and advanced disease in the mixed germ cell tumors. Our findings suggest that DNA methyltransferase 3-like protein may play roles not only in the development of embryonal carcinoma but also in the development of advanced pure seminoma and pure yolk sac tumor. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Production of methoxylated flavonoids in yeast using ring A hydroxylases and flavonoid O-methyltransferases from sweet basil.

PubMed

Berim, Anna; Gang, David R

2018-07-01

Numerous methoxylated flavonoids exhibit pronounced bioactivities. Their biotechnological production and diversification are therefore of interest to pharmaceutical and nutraceutical industries. We used a set of enzymes from sweet basil (Ocimum basilicum) to construct five strains of Saccharomyces cerevisiae producing 8- and/or 6-substituted, methoxylated flavones from their natural precursor apigenin. After identifying several growth parameters affecting the overall yields and flux, we applied optimized conditions and explored the ability of the generated strains to utilize alternative substrates. The yeast cells produced substantial amounts of 6-hydroxylated, methylated derivatives of naringenin and luteolin while the corresponding derivatives of flavonol kaempferol were only detected in trace amounts. Analysis of the intermediates and by-products of the different bioconversions suggested that the substrate specificity of both the hydroxylases and the flavonoid O-methyltransferases is imposing barriers on yields obtained with alternative substrates and highlighted steps that appear to represent bottlenecks en route to increasing the strains' efficiencies. Additionally, analysis of flavonoid localization during fermentation revealed unequal distribution with strong intracellular accumulation of a number of methylated flavonoids and extracellular enrichment of several pathway intermediates. This work establishes a platform for the production of complex methoxylated flavonoids and discusses strategies for its improvement.

Catechol-O-methyltransferase as a target for melanoma destruction?

PubMed

Smit, N P; Latter, A J; Naish-Byfield, S; Westerhof, W; Pavel, S; Riley, P A

1994-08-17

Catechols may interfere in melanogenesis by causing increased levels of toxic quinones. Several catechols and known inhibitors of the enzyme catechol-O-methyltransferase (COMT) were therefore tested for their toxicity towards a pigmented melanoma cell line, UCLA-SO-(M14). The inhibition of thymidine incorporation as a result of exposure to the compounds was measured. All agents were compared to 4-hydroxyanisole (4HA), a depigmenting agent extensively studied as an antimelanoma drug. The compounds were also tested on the epithelial cell line, CNCM-I-(221) in the presence and absence of tyrosinase. All the compounds were more effective than 4HA towards the M14-cells at either 10(-4) M or 10(-5) M. The toxicity of 4HA towards the 221-cells was shown to be completely dependent on the presence of tyrosinase. Effects of the test agents on the 221-cells were also observed in the absence of tyrosinase. Although some of them were shown to be good substrates for tyrosinase only small changes in toxicity were observed as a result of the presence of the enzyme in comparison with 4HA. No direct correlation of the toxicity of the agents and COMT inhibition was observed. The possible mode of action of the compounds through inhibition of COMT and interference in melanogenesis is discussed together with other possibilities and factors involved.

Comparison of 6-mercaptopurine with 6-thioguanine for the analysis of thiopurine S-methyltransferase activity in human erythrocyte by LC-MS/MS.

PubMed

Mei, Shenghui; Li, Xindi; Gong, Xiaoqing; Zhang, Xiaoyi; Li, Xingang; Yang, Li; Zhu, Leting; Zhou, Heng; Liu, Yonghong; Zhou, Anna; Zhang, Xinghu; Zhao, Zhigang

2017-09-01

Thiopurines (TPDs) are first-line drugs in treating neuromyelitis optica spectrum disorders (NMOSD). Evaluation of thiopurine S-methyltransferase activity (TPMT), a major determinant of TPD toxicity, before TPD treatment using 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) as substrate was suggested. However, the equivalent of the two substrates in TPMT activity evaluation was unknown, and an alternative substrate was required in TPMT activity evaluation in patients who were already taking 6-MP or 6-TG. Before evaluating the agreement of 6-MP and 6-TG in TPMT activity measurement in patients with NMOSD, the affinity of the two substrates for the active center of TPMT should be established. A computer-based simulation indicated that 6-MP and 6-TG had similar affinities for the two active sites of TPMT. According to the guidelines, an LC-MS/MS method was developed and validated to evaluate the TPMT activity in human erythrocyte hemolysate using 6-MP or 6-TG as substrates via 1 h incubation at 37°C. The method was applied in 81 patients with NMOSD. Evaluated by Bland-Altman plot, 6-methylmercaptopurine and 6-methylthioguanine represented TPMT activities were in agreement with each other. Further studies are warranted to confirm the results. Copyright © 2017 John Wiley & Sons, Ltd.

Conserved residue lysine165 is essential for the ability of O6-alkylguanine-DNA alkyltransferase to react with O6-benzylguanine.

PubMed Central

Xu-Welliver, M; Kanugula, S; Loktionova, N A; Crone, T M; Pegg, A E

2000-01-01

The role of lysine(165) in the activity of the DNA repair protein, O(6)-alkylguanine-DNA alkyltransferase (AGT), and the ability of AGT to react with the pseudosubstrate inhibitor, O(6)-benzylguanine (BG), was investigated by changing this lysine to all other 19 possibilities. All of these mutants (except for K165T, which could not be tested as it was too poorly active for assay in crude cell extracts) gave BG-resistant AGTs with increases in the amount of inhibitor needed to produce a 50% loss of activity in a 30 min incubation (ED(50)) from 100-fold (K165A) to 2400-fold (K165F). Lys(165) is a completely conserved residue in AGTs from many species, and all of the mutations at this site also reduced the ability to repair methylated DNA. The least deleterious change was that to arginine, which reduced the rate constant for DNA repair by approx. 2.5-fold. Mutant K165R resembled all of the other mutants in being highly resistant to BG, with an ED(50) value for inactivation by BG>200-fold greater than wild-type. Detailed studies of purified K165A AGT showed that the rate constant for repair and the binding to methylated DNA substrates were reduced by 10-20-fold. Despite this, the K165A mutant AGT was able to protect cells from alkylating agents and this protection was not abolished by BG. These results show that, firstly, lysine at position 165 is needed for optimal activity of AGT towards methylated DNA substrates and is essential for efficient reaction with BG; and second, even if the AGT activity towards methylated DNA substrates is impaired by mutations at codon 165, such mutants can protect tumour cells from therapeutic alkylating agents. These results raise the possibility that the conservation of Lys(165) is due to the need for AGT activity towards substrates containing more bulky adducts than O(6)-methylguanine. They also suggest that alterations at Lys(165) may occur during chemotherapy with BG and alkylating agents and could limit the effectiveness of this

Characterization of hMTr1, a Human Cap1 2′-O-Ribose Methyltransferase*

PubMed Central

Bélanger, François; Stepinski, Janusz; Darzynkiewicz, Edward; Pelletier, Jerry

2010-01-01

Cellular eukaryotic mRNAs are capped at their 5′ ends with a 7-methylguanosine nucleotide, a structural feature that has been shown to be important for conferring mRNA stability, stimulating mRNA biogenesis (splicing, poly(A) addition, nucleocytoplasmic transport), and increasing translational efficiency. Whereas yeast mRNAs have no additional modifications to the cap, called cap0, higher eukaryotes are methylated at the 2′-O-ribose of the first or the first and second transcribed nucleotides, called cap1 and cap2, respectively. In the present study, we identify the methyltransferase responsible for cap1 formation in human cells, which we call hMTr1 (also known as FTSJD2 and ISG95). We show in vitro that hMTr1 catalyzes specific methylation of the 2′-O-ribose of the first nucleotide of a capped RNA transcript. Using siRNA-mediated knockdown of hMTr1 in HeLa cells, we demonstrate that hMTr1 is responsible for cap1 formation in vivo. PMID:20713356

Biochemical Characterization and Structural Basis of Reactivity and Regioselectivity Differences between Burkholderia thailandensis and Burkholderia glumae 1,6-Didesmethyltoxoflavin N-Methyltransferase.

PubMed

Fenwick, Michael K; Almabruk, Khaled H; Ealick, Steven E; Begley, Tadhg P; Philmus, Benjamin

2017-08-01

Burkholderia glumae converts the guanine base of guanosine triphosphate into an azapteridine and methylates both the pyrimidine and triazine rings to make toxoflavin. Strains of Burkholderia thailandensis and Burkholderia pseudomallei have a gene cluster encoding seven putative biosynthetic enzymes that resembles the toxoflavin gene cluster. Four of the enzymes are similar in sequence to BgToxBCDE, which have been proposed to make 1,6-didesmethyltoxoflavin (1,6-DDMT). One of the remaining enzymes, BthII1283 in B. thailandensis E264, is a predicted S-adenosylmethionine (SAM)-dependent N-methyltransferase that shows a low level of sequence identity to BgToxA, which sequentially methylates N6 and N1 of 1,6-DDMT to form toxoflavin. Here we show that, unlike BgToxA, BthII1283 catalyzes a single methyl transfer to N1 of 1,6-DDMT in vitro. In addition, we investigated the differences in reactivity and regioselectivity by determining crystal structures of BthII1283 with bound S-adenosylhomocysteine (SAH) or 1,6-DDMT and SAH. BthII1283 contains a class I methyltransferase fold and three unique extensions used for 1,6-DDMT recognition. The active site structure suggests that 1,6-DDMT is bound in a reduced form. The plane of the azapteridine ring system is orthogonal to its orientation in BgToxA. In BthII1283, the modeled SAM methyl group is directed toward the p orbital of N1, whereas in BgToxA, it is first directed toward an sp 2 orbital of N6 and then toward an sp 2 orbital of N1 after planar rotation of the azapteridine ring system. Furthermore, in BthII1283, N1 is hydrogen bonded to a histidine residue whereas BgToxA does not supply an obvious basic residue for either N6 or N1 methylation.

Recurrent high-grade glioma.

PubMed

Quant, Eudocia C; Drappatz, Jan; Wen, Patrick Y; Norden, Andrew D

2010-07-01

Opinions vary on the best treatment options for recurrent high-grade glioma. Some argue that bevacizumab should become standard of care for patients with recurrent glioblastoma, especially in light of recent FDA approval for this indication. However, this opinion is not uniformly accepted. Age, performance status, histology, tumor size and location, O6-methylguanine-DNA methyltransferase (MGMT) methylation status for glioblastoma, 1p/19q status for oligodendroglial tumors, and the number and types of prior therapies are important considerations. In addition, recurrent disease must be distinguished from "pseudoprogression" due to treatment effects. Enrollment in a clinical trial is the optimal choice for most patients with recurrent high-grade glioma after failure of radiation therapy and temozolomide. For patients who are ineligible or do not have access to clinical trials, then either bevacizumab monotherapy or bevacizumab in combination with a second agent such as irinotecan is recommended. Involved-field external beam radiation should be considered for patients with anaplastic gliomas who have not received radiation. For patients with anaplastic astrocytoma who progress after radiotherapy, temozolomide may be used. For patients with anaplastic oligodendroglioma who progress after radiotherapy, PCV chemotherapy and temozolomide are options. Oligodendroglial tumors with 1p/19q deletions are more likely to respond to treatment. In the past, carmustine was commonly used to treat recurrent high-grade glioma, but the utility of carmustine in the modern era is unknown because most studies were performed prior to the widespread use of temozolomide. High-precision re-irradiation such as stereotactic radiosurgery is another option in high-grade glioma, especially for patients with poor bone marrow reserve or inability to tolerate chemotherapy, but there is a paucity of studies with adequate controls. Surgery may be useful as adjuvant treatment for patients with symptoms

Water Deficits Affect Caffeate O-Methyltransferase, Lignification, and Related Enzymes in Maize Leaves. A Proteomic Investigation1[w

PubMed Central

Vincent, Delphine; Lapierre, Catherine; Pollet, Brigitte; Cornic, Gabriel; Negroni, Luc; Zivy, Michel

2005-01-01

Drought is a major abiotic stress affecting all levels of plant organization and, in particular, leaf elongation. Several experiments were designed to study the effect of water deficits on maize (Zea mays) leaves at the protein level by taking into account the reduction of leaf elongation. Proteomic analyses of growing maize leaves allowed us to show that two isoforms of caffeic acid/5-hydroxyferulic 3-O-methyltransferase (COMT) accumulated mostly at 10 to 20 cm from the leaf point of insertion and that drought resulted in a shift of this region of maximal accumulation toward basal regions. We showed that this shift was due to the combined effect of reductions in growth and in total amounts of COMT. Several other enzymes involved in lignin and/or flavonoid synthesis (caffeoyl-CoA 3-O-methyltransferase, phenylalanine ammonia lyase, methylenetetrahydrofolate reductase, and several isoforms of S-adenosyl-l-methionine synthase and methionine synthase) were highly correlated with COMT, reinforcing the hypothesis that the zone of maximal accumulation corresponds to a zone of lignification. According to the accumulation profiles of the enzymes, lignification increases in leaves of control plants when their growth decreases before reaching their final size. Lignin levels analyzed by thioacidolysis confirmed that lignin is synthesized in the region where we observed the maximal accumulation of these enzymes. Consistent with the levels of these enzymes, we found that the lignin level was lower in leaves of plants subjected to water deficit than in those of well-watered plants. PMID:15728345

Production of Two Novel Methoxy-Isoflavones from Biotransformation of 8-Hydroxydaidzein by Recombinant Escherichia coli Expressing O-Methyltransferase SpOMT2884 from Streptomyces peucetius

PubMed Central

Chiang, Chien-Min; Ding, Hsiou-Yu; Tsai, Ya-Ting; Chang, Te-Sheng

2015-01-01

Biotransformation of 8-hydroxydaidzein by recombinant Escherichia coli expressing O-methyltransferase (OMT) SpOMT2884 from Streptomyces peucetius was investigated. Two metabolites were isolated and identified as 7,4′-dihydroxy-8-methoxy-isoflavone (1) and 8,4′-dihydroxy-7-methoxy-isoflavone (2), based on mass, 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectrophotometric analysis. The maximum production yields of compound (1) and (2) in a 5-L fermenter were 9.3 mg/L and 6.0 mg/L, respectively. The two methoxy-isoflavones showed dose-dependent inhibitory effects on melanogenesis in cultured B16 melanoma cells under non-toxic conditions. Among the effects, compound (1) decreased melanogenesis to 63.5% of the control at 25 μM. This is the first report on the 8-O-methylation activity of OMT toward isoflavones. In addition, the present study also first identified compound (1) with potent melanogenesis inhibitory activity. PMID:26610478

Impact of ambient gases on the mechanism of [Cs8Nb6O19]-promoted nerve-agent decomposition.

PubMed

Kaledin, Alexey L; Driscoll, Darren M; Troya, Diego; Collins-Wildman, Daniel L; Hill, Craig L; Morris, John R; Musaev, Djamaladdin G

2018-02-28

The impact of ambient gas molecules (X), NO 2 , CO 2 and SO 2 on the structure, stability and decontamination activity of Cs 8 Nb 6 O 19 polyoxometalate was studied computationally and experimentally. It was found that Cs 8 Nb 6 O 19 absorbs these molecules more strongly than it adsorbs water and Sarin (GB) and that these interactions hinder nerve agent decontamination. The impacts of diamagnetic CO 2 and SO 2 molecules on polyoxoniobate Cs 8 Nb 6 O 19 were fundamentally different from that of NO 2 radical. At ambient temperatures, weak coordination of the first NO 2 radical to Cs 8 Nb 6 O 19 conferred partial radical character on the polyoxoniobate and promoted stronger coordination of the second NO 2 adsorbent to form a stable diamagnetic Cs 8 Nb 6 O 19 /(NO 2 ) 2 species. Moreover, at low temperatures, NO 2 radicals formed stable dinitrogen tetraoxide (N 2 O 4 ) that weakly interacted with Cs 8 Nb 6 O 19 . It was found that both in the absence and presence of ambient gas molecules, GB decontamination by the Cs 8 Nb 6 O 19 species proceeds via general base hydrolysis involving: (a) the adsorption of water and the nerve agent on Cs 8 Nb 6 O 19 /(X), (b) concerted hydrolysis of a water molecule on a basic oxygen atom of the polyoxoniobate and nucleophilic addition of the nascent OH group to the phosphorus center of Sarin, and (c) rapid reorganization of the formed pentacoordinated-phosphorus intermediate, followed by dissociation of either HF or isopropanol and formation of POM-bound isopropyl methyl phosphonic acid (i-MPA) or methyl phosphonofluoridic acid (MPFA), respectively. The presence of the ambient gas molecules increases the energy of the intermediate stationary points relative to the asymptote of the reactants and slightly increases the hydrolysis barrier. These changes closely correlate with the Cs 8 Nb 6 O 19 -X complexation energy. The most energetically stable intermediates of the GB hydrolysis and decontamination reaction were found to be Cs 8 Nb 6 O

A critical re-assessment of DNA repair gene promoter methylation in non-small cell lung carcinoma

PubMed Central

Do, Hongdo; Wong, Nicholas C.; Murone, Carmel; John, Thomas; Solomon, Benjamin; Mitchell, Paul L.; Dobrovic, Alexander

2014-01-01

DNA repair genes that have been inactivated by promoter methylation offer potential therapeutic targets either by targeting the specific repair deficiency, or by synthetic lethal approaches. This study evaluated promoter methylation status for eight selected DNA repair genes (ATM, BRCA1, ERCC1, MGMT, MLH1, NEIL1, RAD23B and XPC) in 56 non-small cell lung cancer (NSCLC) tumours and 11 lung cell lines using the methylation-sensitive high resolution melting (MS-HRM) methodology. Frequent methylation in NEIL1 (42%) and infrequent methylation in ERCC1 (2%) and RAD23B (2%) are reported for the first time in NSCLC. MGMT methylation was detected in 13% of the NSCLCs. Contrary to previous studies, methylation was not detected in ATM, BRCA1, MLH1 and XPC. Data from The Cancer Genome Atlas (TCGA) was consistent with these findings. The study emphasises the importance of using appropriate methodology for accurate assessment of promoter methylation. PMID:24569633

Selection of genetically modified hematopoietic cells in vitro and in vivo using alkylating agent lysomustine.

PubMed

Rozov, F N; Grinenko, T S; Levit, G L; Krasnov, V P; Belyavsky, A V

2010-09-15

Efficient gene transfer into hematopoietic stem cells is vital for the success of gene therapy of hematopoietic and immune system disorders. An in vivo selection system based on a mutant form of the O(6)-methylguanine-DNA-methyltransferase gene (MGMTm) is considered one of the more promising strategies for expansion of hematopoietic cells transduced with viral vectors. Here we demonstrate that MGMTm-expressing cells can be efficiently selected using lysomustine, a nitrosourea derivative of lysine. K562 and murine bone marrow cells expressing MGMTm are protected from the cytotoxic action of lysomustine in vitro. We also show in a murine model that MGMTm-transduced hematopoietic cells can be expanded in vivo on transplantation into sublethally irradiated recipients followed by lysomustine treatment. These results indicate that lysomustine can be used as a potent novel chemoselection drug applicable for gene therapy of hematopoietic and immune system disorders. 2010 Elsevier Inc. All rights reserved.

Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells.

PubMed

Karim, Hazhar; Ghalali, Aram; Lafolie, Pierre; Vitols, Sigurd; Fotoohi, Alan K

2013-07-26

The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1μM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug. Copyright © 2013 Elsevier Inc. All rights reserved.

The catechol-O-methyltransferase gene (COMT) and cognitive function from childhood through adolescence

PubMed Central

Gaysina, Darya; Xu, Man K.; Barnett, Jennifer H.; Croudace, Tim J.; Wong, Andrew; Richards, Marcus; Jones, Peter B.

2013-01-01

Genetic variation in the catechol-O-methyltransferase gene (COMT) can influence cognitive function, and this effect may depend on developmental stage. Using a large representative British birth cohort, we investigated the effect of COMT on cognitive function (verbal and non-verbal) at ages 8 and 15 years taking into account the possible modifying effect of pubertal stage. Five functional COMT polymorphisms, rs6269, rs4818, rs4680, rs737865 and rs165599 were analysed. Associations between COMT polymorphisms and cognition were tested using regression and latent variable structural equation modelling (SEM). Before correction for multiple testing, COMT rs737865 showed association with reading comprehension, verbal ability and global cognition at age 15 years in pubescent boys only. Although there was some evidence for age- and sex-specific effects of the COMT rs737865 none remained significant after correction for multiple testing. Further studies are necessary in order to make firmer conclusions. PMID:23178897

Role of O-methyltransferase in the lignification of Douglas-fir cultured tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monroe, S.H.

1983-01-01

O-methyltransferase (OMT) is a key enzyme in the biosynthesis of lignin. This enzyme was isolated and characterized in an effort to understand why Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) callus tissue does not form appreciable amounts of lignin yet does form large amounts of the related flavonoids and tannins. It was shown that the OMT in the callus tissue is a cell wall associated, membrane-bound enzyme, in contrast to that of all reported plant species and to Douglas-fir seedlings, which have either a microsomal or soluble OMT. The effect this had on the OMT kinetic constants was studied. It was foundmore » that the callus OMT had much higher K/sub m/ constants for caffeic acid in both the membrane-bound and free forms compared with seedlings. The callus membrane-bound K/sub m/ for caffeic acid is 333 ..mu..M. The callus membrane-free K/sub m/ for caffeic acid is 250 ..mu..M. The seedling K/sub m/ for caffeic acid is 90 ..mu..M.« less

Quantum mechanical/molecular mechanical molecular dynamics and free energy simulations of the thiopurine S-methyltransferase reaction with 6-mercaptopurine.

PubMed

Pan, Xiao-Liang; Cui, Feng-Chao; Liu, Jing-Yao

2011-06-23

Quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations were performed to investigate the methylation of 6-mercaptopurine catalyzed by thiopurine S-methyltransferase. Several setups with different tautomeric forms and orientations of the substrate were considered. It is found that, with the orientation in chain A of the X-ray structure, the substrate can form an ideal near-attack configuration for the methylation reaction, which may take place after the deprotonation of the substrate by the conserved residue Asp23 through a water chain. The potential of mean force (PMF) of the methyl-transfer step for the most favorable pathway is 19.6 kcal/mol, which is in good agreement with the available experimental rate constant data.

Protein arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression

PubMed Central

Baldwin, R. Mitchell; Haghandish, Nasim; Daneshmand, Manijeh; Amin, Shahrier; Paris, Geneviève; Falls, Theresa J.; Bell, John C.; Islam, Shahidul; Côté, Jocelyn

2015-01-01

Recent evidence points to the protein arginine methyltransferase (PRMT) family of enzymes playing critical roles in cancer. PRMT7 has been identified in several gene expression studies to be associated with increased metastasis and decreased survival in breast cancer patients. However, this has not been extensively studied. Here we report that PRMT7 expression is significantly upregulated in both primary breast tumour tissues and in breast cancer lymph node metastases. We have demonstrated that reducing PRMT7 levels in invasive breast cancer cells using RNA interference significantly decreased cell invasion in vitro and metastasis in vivo. Conversely, overexpression of PRMT7 in non-aggressive MCF7 cells enhanced their invasiveness. Furthermore, we show that PRMT7 induces the expression of matrix metalloproteinase 9 (MMP9), a well-known mediator of breast cancer metastasis. Importantly, we significantly rescued invasion of aggressive breast cancer cells depleted of PRMT7 by the exogenous expression of MMP9. Our results demonstrate that upregulation of PRMT7 in breast cancer may have a significant role in promoting cell invasion through the regulation of MMP9. This identifies PRMT7 as a novel and potentially significant biomarker and therapeutic target for breast cancer. PMID:25605249

Protein arginine methyltransferase 7 promotes breast cancer cell invasion through the induction of MMP9 expression.

PubMed

Baldwin, R Mitchell; Haghandish, Nasim; Daneshmand, Manijeh; Amin, Shahrier; Paris, Geneviève; Falls, Theresa J; Bell, John C; Islam, Shahidul; Côté, Jocelyn

2015-02-20

Recent evidence points to the protein arginine methyltransferase (PRMT) family of enzymes playing critical roles in cancer. PRMT7 has been identified in several gene expression studies to be associated with increased metastasis and decreased survival in breast cancer patients. However, this has not been extensively studied. Here we report that PRMT7 expression is significantly upregulated in both primary breast tumour tissues and in breast cancer lymph node metastases. We have demonstrated that reducing PRMT7 levels in invasive breast cancer cells using RNA interference significantly decreased cell invasion in vitro and metastasis in vivo. Conversely, overexpression of PRMT7 in non-aggressive MCF7 cells enhanced their invasiveness. Furthermore, we show that PRMT7 induces the expression of matrix metalloproteinase 9 (MMP9), a well-known mediator of breast cancer metastasis. Importantly, we significantly rescued invasion of aggressive breast cancer cells depleted of PRMT7 by the exogenous expression of MMP9. Our results demonstrate that upregulation of PRMT7 in breast cancer may have a significant role in promoting cell invasion through the regulation of MMP9. This identifies PRMT7 as a novel and potentially significant biomarker and therapeutic target for breast cancer.

Interactions among catechol-O-methyltransferase genotype, parenting, and sex predict children’s internalizing symptoms and inhibitory control: Evidence for differential susceptibility

PubMed Central

SULIK, MICHAEL J.; EISENBERG, NANCY; SPINRAD, TRACY L.; LEMERY-CHALFANT, KATHRYN; SWANN, GREGORY; SILVA, KASSONDRA M.; REISER, MARK; STOVER, DARYN A.; VERRELLI, BRIAN C.

2015-01-01

We used sex, observed parenting quality at 18 months, and three variants of the catechol-O-methyltransferase gene (Val158Met [rs4680], intron1 [rs737865], and 3′-untranslated region [rs165599]) to predict mothers’ reports of inhibitory and attentional control (assessed at 42, 54, 72, and 84 months) and internalizing symptoms (assessed at 24, 30, 42, 48, and 54 months) in a sample of 146 children (79 male). Although the pattern for all three variants was very similar, Val158Met explained more variance in both outcomes than did intron1, the 3′-untranslated region, or a haplotype that combined all three catechol-O-methyltransferase variants. In separate models, there were significant three-way interactions among each of the variants, parenting, and sex, predicting the intercepts of inhibitory control and internalizing symptoms. Results suggested that Val158Met indexes plasticity, although this effect was moderated by sex. Parenting was positively associated with inhibitory control for methionine–methionine boys and for valine–valine/valine–methionine girls, and was negatively associated with internalizing symptoms for methionine–methionine boys. Using the “regions of significance” technique, genetic differences in inhibitory control were found for children exposed to high-quality parenting, whereas genetic differences in internalizing were found for children exposed to low-quality parenting. These findings provide evidence in support of testing for differential susceptibility across multiple outcomes. PMID:25159270

Rhabdoid glioblastoma is distinguishable from classical glioblastoma by cytogenetics and molecular genetics.

PubMed

Byeon, Sun-Ju; Cho, Hwa Jin; Baek, Hae Woon; Park, Chul-Kee; Choi, Seung-Hong; Kim, Se-Hoon; Kim, Hee Kyung; Park, Sung-Hye

2014-03-01

The clinicopathologic and molecular genetic features of 5 cases of rhabdoid glioblastoma, an extremely rare variant of glioblastoma that tends to affect patients at a young age, were investigated by immunohistochemical analysis and focused molecular genetic studies including array-based comparative genomic hybridization. All 5 cases had supratentorial tumors that immunohistochemical analysis revealed to be robustly positive for epithelial membrane antigen, vimentin, p53, and PDGFRα (platelet-derived growth factor receptor, alpha polypeptide) but only focally positive for glial fibrillary acidic protein. Although complete retention of SMARCB1 (INI1) was observed in all 5 cases, epidermal growth factor receptor (EGFR) amplification, PTEN (phosphatase and tensin homolog) loss, homozygous deletion of cyclin-dependent kinase inhibitor 2A, 1p/19q codeletion, and isocitrate dehydrogenase 1 R132/IDH2 R172 mutation were not observed in any case, although a high level of EGFR polysomy was detected in 1 recurrent tumor. Although c-MET (MET protein) expression was focal but robustly positive in 3 cases, met proto-oncogene (MET) fluorescence in situ hybridization revealed low polysomy but not MET amplification. MGMT (O-6-methylguanine-DNA methyl-40 transferase) methylation-specific polymerase chain reaction revealed MGMT methylation in only 1 case. Furthermore, array-based comparative genomic hybridization revealed gain of chromosome 7 and loss of 1p, 6, 8p, 11, 13q, and 18q but no deletion of chromosome 22. In contrast to the classical subtype of primary glioblastoma, the cases studied here were characterized by the absence of EGFR amplification, PTEN loss, and 9p homozygous deletion and overexpression of p53, PDGFRα, and c-MET, suggesting that they can be classified as the proneural or mesenchymal subtype of glioblastoma and benefit from intensive therapy that includes temozolomide. Copyright © 2014 Elsevier Inc. All rights reserved.

Clinical and Genetic Factors Associated With Severe Hematological Toxicity in Glioblastoma Patients During Radiation Plus Temozolomide Treatment: A Prospective Study.

PubMed

Lombardi, Giuseppe; Rumiato, Enrica; Bertorelle, Roberta; Saggioro, Daniela; Farina, Patrizia; Della Puppa, Alessandro; Zustovich, Fable; Berti, Franco; Sacchetto, Valeria; Marcato, Raffaella; Amadori, Alberto; Zagonel, Vittorina

2015-10-01

Temozolomide (TMZ) administered daily with radiation therapy (RT) for 6 weeks, followed by adjuvant TMZ for 6 cycles, is the standard therapy for newly diagnosed glioblastoma (GBM) patients. Although TMZ is considered to be a safe drug, it has been demonstrated to cause severe myelotoxicity; in particular, some case reports and small series studies have reported severe myelotoxicity developing during TMZ and concomitant RT. We performed a prospective study to analyze the incidence of early severe myelotoxicity and its possible clinical and genetic factors. From November 2010 to July 2012, newly diagnosed GBM patients were enrolled. They were eligible for the study if they met the following criteria: pathologically proven GBM, age 18 years and older, an Eastern Cooperative Oncology Group performance status of 0 to 2, adequate renal and hepatic function, and adequate blood cell counts before starting TMZ plus RT. Grading of hematologic toxicity developing during radiation and TMZ was based on the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0. Clinical factors from all patients were recorded. The methylation status and polymorphic variants of O-methylguanine-DNAmethyl-transferase gene in peripheral blood mononuclear cells, and polymorphic genetic variants of genes involved in the pharmacokinetics and pharmacodynamics of TMZ, were analyzed. For genetic analyses, patients with toxicity were matched (1:2) for age, performance status, anticonvulsants, and proton pump inhibitors with patients without myelotoxicity. We enrolled 87 consecutive GBM patients: 32 women and 55 men; the average age was 60 years. During TMZ and RT, 4 patients (5%) showed grade 3-4 myelotoxicity, and its median duration was 255 days. Predictor factors of severe myelotoxicity were female sex, pretreatment platelet count of ≤3,00,000/mm, methylated O-methylguanine-DNA methyltransferase promoter in the hematopoietic cell system, and specific polymorphic

S-adenosylmethionine regulates thiopurine methyltransferase activity and decreases 6-mercaptopurine cytotoxicity in MOLT lymphoblasts.

PubMed

Milek, Miha; Karas Kuzelicki, Natasa; Smid, Alenka; Mlinaric-Rascan, Irena

2009-06-15

Six-mercaptopurine (6-MP) is a pro-drug widely used in treatment of various diseases, including acute lymphoblastic leukaemia (ALL). Side-effects of thiopurine therapy have been correlated with thiopurine methyltransferase (TPMT) activity. We propose a novel TPMT-mediated mechanism of S-adenosylmethionine (SAM)-specific effects on 6-mercaptopurine (6-MP) induced cytotoxicity in a model cell line for acute lymphoblastic leukemia (MOLT). Our results show that exogenous SAM (10-50microM) rescues cells from the toxic effects of 6-MP (5microM) by delaying the onset of apoptosis. We prove that the extent of methylthioinosine monophosphate (MeTIMP) induced inhibition of de novo purine synthesis (DNPS) determines the concentrations of intracellular ATP, and consequently SAM, which acts as a positive modulator of TPMT activity. This leads to a greater conversion of 6-MP to inactive 6-methylmercaptopurine, and thus lower availability of thioinosine monophosphate for the biotransformation to cytotoxic thioguanine nucleotides (TGNs) and MeTIMP. We further show that the addition of exogenous SAM to 6-MP treated cells maintains intracellular SAM levels, TPMT activity and protein levels, all of which are diminished in cells incubated with 6-MP. Since TPMT mRNA levels remained unaltered, the effect of SAM appears to be restricted to protein stabilisation rather than an increase of TPMT expression. We thus propose that SAM reverses the extent of 6-MP cytotoxicity, by acting as a TPMT-stabilizing factor. This study provides new insights into the pharmacogenetics of thiopurine drugs. Identification of SAM as critical modulator of TPMT activity and consequently thiopurine toxicity may set novel grounds for the rationalization of thiopurine therapy.

Catechol-O-Methyltransferase and UDP-Glucuronosyltransferases in the Metabolism of Baicalein in Different Species.

PubMed

Zhang, Ruiya; Cui, Yonglei; Wang, Yan; Tian, Xiangge; Zheng, Lu; Cong, HaiJian; Wu, Bin; Huo, Xiaokui; Wang, Chao; Zhang, BaoJing; Wang, Xiaobo; Yu, Zhonghui

2017-12-01

Baicalein is the major bioactive flavonoid in some herb medicines and dietary plants; however, the detailed metabolism pathway of its major metabolite oroxylin A-7-O-β-D-glucuronide in human was not clear. It was important to illustrate the major metabolic enzymes that participate in its elimination for the clinic use of baicalein. We first revealed a two-step metabolism profile for baicalein and illustrated the combination of catechol-O-methyltransferase (COMT) and uridine diphosphate-glucuronosyltransferases (UGTs) in drug metabolism, further evaluated its bioactivity variation during drug metabolism. The metabolism profiles were systematically characterized in different human biology preparations; after then, the anti-inflammatory activities of metabolites were evaluated in LPS-induced RAW264.7 cell. The first-step metabolite of baicalein was isolated and identified as oroxylin A; soluble-bound COMT (S-COMT) was the major enzyme responsible for its biotransformation. Specially, position 108 mutation of S-COMT significantly decreases the elimination. Meantime, oroxylin A was rapidly metabolized by UGTs, UGT1A1, -1A3, -1A6, -1A7, -1A8, -1A9, and -1A10 which were involved in the glucuronidation. Considerable species differences were observed with 1060-fold K m (3.05 ± 1.86-3234 ± 475 μM) and 330-fold CL int (5.93-1973 μL/min/mg) variations for baicalein metabolism. Finally, the middle metabolite oroxylin A exhibited a potent anti-inflammatory activity with the IC 50 value of 28 μM. The detailed kinetic parameters indicated that COMT provide convenience for the next glucuronidation; monkey would be a preferred animal model for the preclinical investigation of baicalein. Importantly, oroxylin A should be reconsidered in evaluating baicalein efficacy against inflammatory diseases.

Influence of dioxygen on the promotional effect of Bi during Pt-catalyzed oxidation of 1,6-hexanediol

DOE PAGES

Xie, Jiahan; Huang, Benjamin; Yin, Kehua; ...

2016-05-24

In this study, a series of carbon-supported, Bi-promoted Pt catalysts with various Bi/Pt atomic ratios was prepared by selectively depositing Bi on Pt nanoparticles. The catalysts were evaluated for 1,6-hexanediol oxidation activity in aqueous solvent under different dioxygen pressures. The rate of diol oxidation on the basis of Pt loading over a Bi-promoted catalyst was 3 times faster than that of an unpromoted Pt catalyst under 0.02 MPa of O 2, whereas the unpromoted catalyst was more active than the promoted catalyst under 1 MPa of O 2. After liquid-phase catalyst pretreatment and 1,6-hexanediol oxidation, migration of Bi on themore » carbon support was observed. The reaction order in O 2 was 0 over Bi-promoted Pt/C in comparison to 0.75 over unpromoted Pt/C in the range of 0.02–0.2 MPa of O 2. Under low O 2 pressure, rate measurements in D 2O instead of H 2O solvent revealed a moderate kinetic isotope effect (rate H2O/rate D2O) on 1,6-hexanediol oxidation over Pt/C (KIE = 1.4), whereas a negligible effect was observed on Bi-Pt/C (KIE = 0.9), indicating that the promotional effect of Bi could be related to the formation of surface hydroxyl groups from the reaction of dioxygen and water. No significant change in product distribution or catalyst stability was observed with Bi promotion, regardless of the dioxygen pressure.« less

Age-Dependent Effects of Catechol-O-Methyltransferase (COMT) Gene Val158Met Polymorphism on Language Function in Developing Children.

PubMed

Sugiura, Lisa; Toyota, Tomoko; Matsuba-Kurita, Hiroko; Iwayama, Yoshimi; Mazuka, Reiko; Yoshikawa, Takeo; Hagiwara, Hiroko

2017-01-01

The genetic basis controlling language development remains elusive. Previous studies of the catechol-O-methyltransferase (COMT) Val158Met genotype and cognition have focused on prefrontally guided executive functions involving dopamine. However, COMT may further influence posterior cortical regions implicated in language perception. We investigated whether COMT influences language ability and cortical language processing involving the posterior language regions in 246 children aged 6-10 years. We assessed language ability using a language test and cortical responses recorded during language processing using a word repetition task and functional near-infrared spectroscopy. The COMT genotype had significant effects on language performance and processing. Importantly, Met carriers outperformed Val homozygotes in language ability during the early elementary school years (6-8 years), whereas Val homozygotes exhibited significant language development during the later elementary school years. Both genotype groups exhibited equal language performance at approximately 10 years of age. Val homozygotes exhibited significantly less cortical activation compared with Met carriers during word processing, particularly at older ages. These findings regarding dopamine transmission efficacy may be explained by a hypothetical inverted U-shaped curve. Our findings indicate that the effects of the COMT genotype on language ability and cortical language processing may change in a narrow age window of 6-10 years. © The Author 2016. Published by Oxford University Press.

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE PAGES

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh; ...

2015-02-18

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

PubMed

Gonzalo, Victoria; Lozano, Juan José; Muñoz, Jenifer; Balaguer, Francesc; Pellisé, Maria; Rodríguez de Miguel, Cristina; Andreu, Montserrat; Jover, Rodrigo; Llor, Xavier; Giráldez, M Dolores; Ocaña, Teresa; Serradesanferm, Anna; Alonso-Espinaco, Virginia; Jimeno, Mireya; Cuatrecasas, Miriam; Sendino, Oriol; Castellví-Bel, Sergi; Castells, Antoni

2010-01-19

Colorectal cancer (CRC) multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals) and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2), RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008) and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047) as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006). Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17), SFRP1 (r = 0.83, 0.06), HPP1 (r = 0.64, p = 0.17), 3OST2 (r = 0.83, p = 0.06) and GATA4 (r = 0.6, p = 0.24). Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant difference in any evaluated gene. These results provide

The Flexible Mind Is Associated with the Catechol-O-Methyltransferase (COMT) Val[superscript 158]Met Polymorphism: Evidence for a Role of Dopamine in the Control of Task-Switching

ERIC Educational Resources Information Center

Colzato, Lorenza S.; Waszak, Florian; Nieuwenhuis, Sander; Posthuma, Danielle; Hommel, Bernhard

2010-01-01

Genetic variability related to the catechol-O-methyltransferase (COMT) gene Val[superscript 128]Met polymorphism) has received increasing attention as a possible modulator of cognitive control functions. Recent evidence suggests that the Val[superscript 128]Met genotype may differentially affect cognitive stability and flexibility, in such a way…

Genetic markers predictive of chemosensitivity and outcome in gliomatosis cerebri.

PubMed

Kaloshi, G; Everhard, S; Laigle-Donadey, F; Marie, Y; Navarro, S; Mokhtari, K; Idbaih, A; Ducray, F; Thillet, J; Hoang-Xuan, K; Delattre, J-Y; Sanson, M

2008-02-19

Up-front temozolomide (TMZ) has been recently proposed as a treatment for gliomatosis cerebri (GC), but no predictive or prognostic markers have been identified so far. Because 1p19q codeletion and methylguanine methyl transferase promoter (MGMTP) methylation have been correlated with chemosensitivity of gliomas, their value was investigated in a cohort of patients with GC treated with TMZ. A cohort of 25 GC patients who were treated with TMZ was investigated for 1p19q codeletion and O6-methylguanine DNA. Patients with a 1p/19q codeletion had a higher response rate (88% [8/9] vs 25% [4/16], p = 0.002), higher progression-free survival (24.5 vs 13.7 months, p = 0.017), and higher overall survival (66.8 vs 15.2 months, p = 0.011) than patients without 1p/19q codeletion. Fourteen of 19 evaluable tumors for MGMTP status were methylated. MGMTP methylation was associated with 1p/19q codeletion (p = 0.045). Patients with unmethylated MGMTP tended to have a shorter progression-free survival and a higher rate of progressive disease. Response rate to temozolomide and prognosis seem tightly correlated to 1p19q loss. The impact of methylguanine methyl transferase promoter methylation status on gliomatosis cerebri is still unsettled in this population.

Automethylation of protein arginine methyltransferase 7 and its impact on breast cancer progression.

PubMed

Geng, Pengyu; Zhang, Yu; Liu, Xiaoqing; Zhang, Na; Liu, Yingqi; Liu, Xin; Lin, Cong; Yan, Xu; Li, Zhongwei; Wang, Guannan; Li, Yuxin; Tan, Jiang; Liu, Dong-Xu; Huang, Baiqu; Lu, Jun

2017-06-01

Protein arginine methyltransferases (PRMTs) catalyze protein arginine methylation and are linked to carcinogenesis and metastasis. Some members of PRMTs have been found to undergo automethylation; however, the biologic significance of this self-modification is not entirely clear. In this report, we demonstrate that R531 of PRMT7 is self-methylated, both in vitro and in vivo Automethylation of PRMT7 plays a key role in inducing the epithelial-mesenchymal transition (EMT) program and in promoting the migratory and invasive behavior of breast cancer cells. We also prove in a nude mouse model that expression of wild-type PRMT7 in MCF7 breast cancer cells promotes metastasis in vivo , in contrast to the PRMT7 R531K mutant (a mimic of the unmethylated status). Moreover, our immunohistochemical data unravel a close link between PRMT7 automethylation and the clinical outcome of breast carcinomas. Mechanistically, we determine that loss of PRMT7 automethylation leads to the reduction of its recruitment to the E-cadherin promoter by YY1, which consequently derepresses the E-cadherin expression through decreasing the H4R3me2s level. The findings in this work define a novel post-translational modification of PRMT7 that has a promoting impact on breast cancer metastasis.-Geng, P., Zhang, Y., Liu, X., Zhang, N., Liu, Y., Liu, X., Lin, C., Yan, X., Li, Z., Wang, G., Li, Y., Tan, J., Liu, D.-X., Huang, B., Lu, J. Automethylation of protein arginine methyltransferase 7 and its impact on breast cancer progression. © FASEB.

Advances in Translational Research in Neuro-oncology

PubMed Central

Fueyo, Juan; Gomez-Manzano, Candelaria; Yung, W. K. Alfred

2011-01-01

During the last decade, we have witnessed several key advances in the field of neurooncology. First, there were conceptual advances in the molecular and cell biology of malignant gliomas including the discovery in 2004 of brain tumor stem cells. Second, the Cancer Genome Atlas project has been extremely useful in the discovery of new molecular markers, including mutations in the IDH1 gene, and has led to a new classification of gliomas based on the differentiation status and mesenchymal transformation. In addition, use of the 1p/19q marker and O6-methylguanine-DNA methyltransferase methylation status have been identified as guides for patient selection for therapies and represent the first steps toward personalized medicine for treating gliomas. Finally, progress has been made in treatment strategies including the establishment of temozolomide as the criterion standard for treating gliomas, the adoption of bevacizumab in the clinical setting, and developments in experimental biological therapies including cancer vaccines and oncolytic adenoviruses. PMID:21059986

Advances in translational research in neuro-oncology.

PubMed

Fueyo, Juan; Gomez-Manzano, Candelaria; Yung, W K Alfred

2011-03-01

During the last decade, we have witnessed several key advances in the field of neuro-oncology. First, there were conceptual advances in the molecular and cell biology of malignant gliomas including the discovery in 2004 of brain tumor stem cells. Second, the Cancer Genome Atlas project has been extremely useful in the discovery of new molecular markers, including mutations in the IDH1 gene, and has led to a new classification of gliomas based on the differentiation status and mesenchymal transformation. In addition, use of the 1p/19q marker and O6-methylguanine-DNA methyltransferase methylation status have been identified as guides for patient selection for therapies and represent the first steps toward personalized medicine for treating gliomas. Finally, progress has been made in treatment strategies including the establishment of temozolomide as the criterion standard for treating gliomas, the adoption of bevacizumab in the clinical setting, and developments in experimental biological therapies including cancer vaccines and oncolytic adenoviruses.

Gliomatosis cerebri type II: two case reports

PubMed Central

D’Urso, Pietro Ivo; Marsigliante, Santo; Storelli, Carlo; Distante, Alessandro; Sanguedolce, Francesca; Cimmino, Antonia; Luzi, Giuseppe; Gianfreda, Cosimo Damiano; Montinaro, Antonio; Ciappetta, Pasqualino

2009-01-01

Introduction Two types of gliomatosis cerebri exist: Type I and Type II. We report the results of a histological and genetic study of two cases of gliomatosis cerebri Type II, correlating these results with therapy and prognosis. Case presentation Two patients, a 52-year-old man (Patient 1) and a 76-year-old man (Patient 2) with gliomatosis cerebri II were admitted to our institution; they underwent surgical treatment and received radiotherapy and chemotherapy. At the 24-month follow-up, Patient 1 was still alive, while Patient 2 had died. The poor prognosis of Patient 2 was underlined by molecular analysis which showed that the angiogenesis related genes VCAM1 and VEGF were overexpressed, reflecting the high degree of neovascularization. Conclusion Genes involved in drug resistance and metallothioneins were highly expressed in Patient 2 and this, associated with unmethylated O6-methylguanine methyltransferase, can explain the lack of response to chemotherapy. PMID:19830138

Elicitor-Induced Association of Isoflavone O-Methyltransferase with Endomembranes Prevents the Formation and 7-O-Methylation of Daidzein during Isoflavonoid Phytoalexin Biosynthesis

PubMed Central

Liu, Chang-Jun; Dixon, Richard A.

2001-01-01

The bioactive isoflavonoids of the Leguminosae often are methylated on the 4′-position of their B-rings. Paradoxically, reverse genetic evidence implicates alfalfa isoflavone O-methyltransferase (IOMT) in the biosynthesis of 4′-O-methylated isoflavonoids such as the phytoalexin medicarpin in vivo, whereas biochemical studies indicate that IOMT has strict specificity for methylation of the A-ring 7-hydroxyl of daidzein, the presumed substrate for O-methylation, in vitro. Radiolabeling and isotope dilution studies now confirm that daidzein is not an intermediate in isoflavonoid phytoalexin biosynthesis in alfalfa. Furthermore, protein gel blot analysis and confocal microscopy of a transiently expressed IOMT–green fluorescent protein fusion in alfalfa leaves show that the operationally soluble IOMT localizes to endomembranes after elicitation of the isoflavonoid pathway. We propose that IOMT colocalizes with the endoplasmic reticulum–associated isoflavone synthase cytochrome P450 to ensure rapid B-ring methylation of the unstable 2,4′,7-trihydroxyisoflavanone product of isoflavone synthase, thereby preventing its dehydration to daidzein and subsequent A-ring methylation by free IOMT. In this way, metabolic channeling at the entry point into isoflavonoid phytoalexin biosynthesis protects an unstable intermediate from an unproductive metabolic conversion. PMID:11752378

Molecular Mapping of the RNA Cap 2′-O-Methyltransferase Activation Interface between Severe Acute Respiratory Syndrome Coronavirus nsp10 and nsp16*

PubMed Central

Lugari, Adrien; Betzi, Stephane; Decroly, Etienne; Bonnaud, Emmanuel; Hermant, Aurélie; Guillemot, Jean-Claude; Debarnot, Claire; Borg, Jean-Paul; Bouvet, Mickaël; Canard, Bruno; Morelli, Xavier; Lécine, Patrick

2010-01-01

Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several “hot spots,” such as Val42, Met44, Ala71, Lys93, Gly94, and Tyr96, forming a continuous protein-protein surface of about 830 Å2, bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2′-O-methyltransferase (2′O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2′O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2′O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses. PMID:20699222

Carmustine wafer implantation for high-grade gliomas: Evidence-based safety efficacy and practical recommendations from the Neuro-oncology Club of the French Society of Neurosurgery.

PubMed

Roux, A; Caire, F; Guyotat, J; Menei, P; Metellus, P; Pallud, J

2017-12-01

There is a growing body of evidence that carmustine wafer implantation during surgery is an effective therapeutic adjunct to the standard combined radio-chemotherapy regimen using temozolomide in newly diagnosed and recurrent high-grade glioma patient management with a statistically significant survival benefit demonstrated across several randomized clinical trials, as well as prospective and retrospective studies (grade A recommendation). Compelling clinical data also support the safety of carmustine wafer implantation (grade A recommendation) in these patients and suggest that observed adverse events can be avoided in experienced neurosurgeon hands. Furthermore, carmustine wafer implantation does not seem to impact negatively on the quality of life and the completion of adjuvant oncological treatments (grade C recommendation). Moreover, emerging findings support the potential of high-grade gliomas molecular status, especially the O(6)-Methylguanine-DNA Methyltransferase promoter methylation status, in predicting the efficacy of such a surgical strategy, especially at recurrence (grade B recommendation). Finally, carmustine wafer implantation appears to be cost-effective in high-grade glioma patients when performed by an experienced team and when total or subtotal resection can be achieved. Altogether, these data underline the current need for a new randomized clinical trial to assess the impact of a maximal safe resection with carmustine wafer implantation followed by the standard combined chemoradiation protocol stratified by molecular status in high-grade glioma patients. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Large Animal Models for Foamy Virus Vector Gene Therapy

PubMed Central

Trobridge, Grant D.; Horn, Peter A.; Beard, Brian C.; Kiem, Hans-Peter

2012-01-01

Foamy virus (FV) vectors have shown great promise for hematopoietic stem cell (HSC) gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit. PMID:23223198

HDA6 directly interacts with DNA methyltransferase MET1 and maintains transposable element silencing in Arabidopsis.

PubMed

Liu, Xuncheng; Yu, Chun-Wei; Duan, Jun; Luo, Ming; Wang, Koching; Tian, Gang; Cui, Yuhai; Wu, Keqiang

2012-01-01

The molecular mechanism of how the histone deacetylase HDA6 participates in maintaining transposable element (TE) silencing in Arabidopsis (Arabidopsis thaliana) is not yet defined. In this study, we show that a subset of TEs was transcriptionally reactivated and that TE reactivation was associated with elevated histone H3 and H4 acetylation as well as increased H3K4Me3 and H3K4Me2 in hda6 mutants. Decreased DNA methylation of the TEs was also detected in hda6 mutants, suggesting that HDA6 silences the TEs by regulating histone acetylation and methylation as well as the DNA methylation status of the TEs. Similarly, transcripts of some of these TEs were also increased in the methyltransferase1 (met1) mutant, with decreased DNA methylation. Furthermore, H4 acetylation, H3K4Me3, H3K4Me2, and H3K36Me2 were enriched at the coregulated TEs in the met1 and hda6 met1 mutants. Protein-protein interaction analysis indicated that HDA6 physically interacts with MET1 in vitro and in vivo, and further deletion analysis demonstrated that the carboxyl-terminal region of HDA6 and the bromo-adjacent homology domain of MET1 were responsible for the interaction. These results suggested that HDA6 and MET1 interact directly and act together to silence TEs by modulating DNA methylation, histone acetylation, and histone methylation status.

Expression of exogenous DNA methyltransferases: application in molecular and cell biology.

PubMed

Dyachenko, O V; Tarlachkov, S V; Marinitch, D V; Shevchuk, T V; Buryanov, Y I

2014-02-01

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.

A vicious loop of fatty acid-binding protein 4 and DNA methyltransferase 1 promotes acute myeloid leukemia and acts as a therapeutic target

PubMed Central

Yan, F; Shen, N; Pang, JX; Zhao, N; Zhang, YW; Bode, AM; Al-Kali, A; Litzow, MR; Li, B; Liu, SJ

2017-01-01

Aberrant DNA methylation mediated by deregulation of DNA methyltransferases (DNMT) is a key hallmark of acute myeloid leukemia (AML), yet efforts to target DNMT deregulation for drug development have lagged. We previously demonstrated that upregulation of fatty acid-binding protein 4 (FABP4) promotes AML aggressiveness through enhanced DNMT1-dependent DNA methylation. Here we demonstrate that FABP4 upregulation in AML cells occurs through vascular endothelial growth factor (VEGF) signaling, thus elucidating a crucial FABP4-DNMT1 regulatory feedback loop in AML biology. We show that FABP4 dysfunction by its selective inhibitor BMS309403 leads to downregulation of DNMT1, decrease of global DNA methylation and re-expression of p15INK4B tumor suppressor gene by promoter DNA hypomethylation in vitro, ex vivo and in vivo. Functionally, BMS309403 suppresses cell colony formation, induces cell differentiation, and, importantly, impairs leukemic disease progression in mouse models of leukemia. Our findings highlight AML-promoting properties of the FABP4-DNMT1 vicious loop, and identify an attractive class of therapeutic agents with a high potential for clinical use in AML patients. The results will also assist in establishing the FABP4-DNMT1 loop as a target for therapeutic discovery to enhance the index of current epigenetic therapies. PMID:28993705

An Iterative O-Methyltransferase Catalyzes 1,11-Dimethylation of Aspergillus fumigatus Fumaric Acid Amides.

PubMed

Kalb, Daniel; Heinekamp, Thorsten; Schieferdecker, Sebastian; Nett, Markus; Brakhage, Axel A; Hoffmeister, Dirk

2016-10-04

S-adenosyl-l-methionine (SAM)-dependent methyltransfer is a common biosynthetic strategy to modify natural products. We investigated the previously uncharacterized Aspergillus fumigatus methyltransferase FtpM, which is encoded next to the bimodular fumaric acid amide synthetase FtpA. Structure elucidation of two new A. fumigatus natural products, the 1,11-dimethyl esters of fumaryl-l-tyrosine and fumaryl-l-phenylalanine, together with ftpM gene disruption suggested that FtpM catalyzes iterative methylation. Final evidence that a single enzyme repeatedly acts on fumaric acid amides came from an in vitro biochemical investigation with recombinantly produced FtpM. Size-exclusion chromatography indicated that this methyltransferase is active as a dimer. As ftpA and ftpM homologues are found clustered in other fungi, we expect our work will help to identify and annotate natural product biosynthesis genes in various species. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DNA (Cytosine-C5) methyltransferase inhibition by oligodeoxyribonucleotides containing 2-(1H)-pyrimidinone (zebularine aglycon) at the enzymatic target site.

PubMed

van Bemmel, Dana M; Brank, Adam S; Eritja, Ramon; Marquez, Victor E; Christman, Judith K

2009-09-15

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors.

DNA (Cytosine-C5) Methyltransferase Inhibition by Oligodeoxyribonucleotides Containing 2-(1H)-Pyrimidinone (Zebularine Aglycon) at the Enzymatic Target Site

PubMed Central

van Bemmel, Dana M.; Brank, Adam S.; Eritja, Ramon; Marquez, Victor E.; Christman, Judith K.

2009-01-01

Aberrant cytosine methylation in promoter regions leads to gene silencing associated with cancer progression. A number of DNA methyltransferase inhibitors are known to reactivate silenced genes; including 5-azacytidine and 2-(1H)-pyrimidinone riboside (zebularine). Zebularine is a more stable, less cytotoxic inhibitor compared to 5-azacytidine. To determine the mechanistic basis for this difference, we carried out a detailed comparisons of the interaction between purified DNA methyltransferases and oligodeoxyribonucleotides (ODNs) containing either 5-azacytosine or 2-(1H)-pyrimidinone in place of the cytosine targeted for methylation. When incorporated into small ODNs, the rate of C5 DNA methyltransferase inhibition by both nucleosides is essentially identical. However, the stability and reversibility of the enzyme complex in the absence and presence of cofactor differs. 5-Azacytosine ODNs form complexes with C5 DNA methyltransferases that are irreversible when the 5-azacytosine ring is intact. ODNs containing 2-(1H)-pyrimidinone at the enzymatic target site are competitive inhibitors of both prokaryotic and mammalian DNA C5 methyltransferases. We determined that the ternary complexes between the enzymes, 2-(1H)-pyrimidinone inhibitor, and the cofactor S-adenosyl methionine are maintained through the formation of a reversible covalent interaction. The differing stability and reversibility of the covalent bonds may partially account for the observed differences in cytotoxicity between zebularine and 5-azacytidine inhibitors. PMID:19467223

Persistent Catechol-O-methyltransferase-dependent Pain Is Initiated by Peripheral β-Adrenergic Receptors.

PubMed

Ciszek, Brittney P; O'Buckley, Sandra C; Nackley, Andrea G

2016-05-01

Patients with chronic pain disorders exhibit increased levels of catecholamines alongside diminished activity of catechol-O-methyltransferase (COMT), an enzyme that metabolizes catecholamines. The authors found that acute pharmacologic inhibition of COMT in rodents produces hypersensitivity to mechanical and thermal stimuli via β-adrenergic receptor (βAR) activation. The contribution of distinct βAR populations to the development of persistent pain linked to abnormalities in catecholamine signaling requires further investigation. Here, the authors sought to determine the contribution of peripheral, spinal, and supraspinal βARs to persistent COMT-dependent pain. They implanted osmotic pumps to deliver the COMT inhibitor OR486 (Tocris, USA) for 2 weeks. Behavioral responses to mechanical and thermal stimuli were evaluated before and every other day after pump implantation. The site of action was evaluated in adrenalectomized rats receiving sustained OR486 or in intact rats receiving sustained βAR antagonists peripherally, spinally, or supraspinally alongside OR486. The authors found that male (N = 6) and female (N = 6) rats receiving sustained OR486 exhibited decreased paw withdrawal thresholds (control 5.74 ± 0.24 vs. OR486 1.54 ± 0.08, mean ± SEM) and increased paw withdrawal frequency to mechanical stimuli (control 4.80 ± 0.22 vs. OR486 8.10 ± 0.13) and decreased paw withdrawal latency to thermal heat (control 9.69 ± 0.23 vs. OR486 5.91 ± 0.11). In contrast, adrenalectomized rats (N = 12) failed to develop OR486-induced hypersensitivity. Furthermore, peripheral (N = 9), but not spinal (N = 4) or supraspinal (N = 4), administration of the nonselective βAR antagonist propranolol, the β2AR antagonist ICI-118,511, or the β3AR antagonist SR59230A blocked the development of OR486-induced hypersensitivity. Peripheral adrenergic input is necessary for the development of persistent COMT-dependent pain, and peripherally-acting βAR antagonists may benefit

N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection.

PubMed

Gokhale, Nandan S; McIntyre, Alexa B R; McFadden, Michael J; Roder, Allison E; Kennedy, Edward M; Gandara, Jorge A; Hopcraft, Sharon E; Quicke, Kendra M; Vazquez, Christine; Willer, Jason; Ilkayeva, Olga R; Law, Brittany A; Holley, Christopher L; Garcia-Blanco, Mariano A; Evans, Matthew J; Suthar, Mehul S; Bradrick, Shelton S; Mason, Christopher E; Horner, Stacy M

2016-11-09

The RNA modification N6-methyladenosine (m 6 A) post-transcriptionally regulates RNA function. The cellular machinery that controls m 6 A includes methyltransferases and demethylases that add or remove this modification, as well as m 6 A-binding YTHDF proteins that promote the translation or degradation of m 6 A-modified mRNA. We demonstrate that m 6 A modulates infection by hepatitis C virus (HCV). Depletion of m 6 A methyltransferases or an m 6 A demethylase, respectively, increases or decreases infectious HCV particle production. During HCV infection, YTHDF proteins relocalize to lipid droplets, sites of viral assembly, and their depletion increases infectious viral particles. We further mapped m 6 A sites across the HCV genome and determined that inactivating m 6 A in one viral genomic region increases viral titer without affecting RNA replication. Additional mapping of m 6 A on the RNA genomes of other Flaviviridae, including dengue, Zika, yellow fever, and West Nile virus, identifies conserved regions modified by m 6 A. Altogether, this work identifies m 6 A as a conserved regulatory mark across Flaviviridae genomes. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

How Large Should the QM Region Be in QM/MM Calculations? The Case of Catechol O -Methyltransferase

DOE PAGES

Kulik, Heather J.; Zhang, Jianyu; Klinman, Judith P.; ...

2016-10-05

Hybrid quantum mechanical–molecular mechanical (QM/MM) simulations are widely used in studies of enzymatic catalysis. Until recently, it has been cost prohibitive to determine the asymptotic limit of key energetic and structural properties with respect to increasingly large QM regions. Here, leveraging recent advances in electronic structure efficiency and accuracy, we investigate catalytic properties in catechol O-methyltransferase, a prototypical methyltransferase critical to human health. Using QM regions ranging in size from reactants-only (64 atoms) to nearly one-third of the entire protein (940 atoms), we show that properties such as the activation energy approach within chemical accuracy of the large-QM asymptotic limitsmore » rather slowly, requiring approximately 500–600 atoms if the QM residues are chosen simply by distance from the substrate. This slow approach to asymptotic limit is due to charge transfer from protein residues to the reacting substrates. Our large QM/MM calculations enable identification of charge separation for fragments in the transition state as a key component of enzymatic methyl transfer rate enhancement. We introduce charge shift analysis that reveals the minimum number of protein residues (approximately 11–16 residues or 200–300 atoms for COMT) needed for quantitative agreement with large-QM simulations. The identified residues are not those that would be typically selected using criteria such as chemical intuition or proximity. These results provide a recipe for a more careful determination of QM region sizes in future QM/MM studies of enzymes.« less

Inhibition of mitogen-activated protein kinase kinase, DNA methyltransferase, and transforming growth factor-β promotes differentiation of human induced pluripotent stem cells into enterocytes.

PubMed

Kodama, Nao; Iwao, Takahiro; Kabeya, Tomoki; Horikawa, Takashi; Niwa, Takuro; Kondo, Yuki; Nakamura, Katsunori; Matsunaga, Tamihide

2016-06-01

We previously reported that small-molecule compounds were effective in generating pharmacokinetically functional enterocytes from human induced pluripotent stem (iPS) cells. In this study, to determine whether the compounds promote the differentiation of human iPS cells into enterocytes, we investigated the effects of a combination of mitogen-activated protein kinase kinase (MEK), DNA methyltransferase (DNMT), and transforming growth factor (TGF)-β inhibitors on intestinal differentiation. Human iPS cells cultured on feeder cells were differentiated into endodermal cells by activin A. These endodermal-like cells were then differentiated into intestinal stem cells by fibroblast growth factor 2. Finally, the cells were differentiated into enterocyte cells by epidermal growth factor and small-molecule compounds. After differentiation, mRNA expression levels and drug-metabolizing enzyme activities were measured. The mRNA expression levels of the enterocyte marker sucrase-isomaltase and the major drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 were increased by a combination of MEK, DNMT, and TGF-β inhibitors. The mRNA expression of CYP3A4 was markedly induced by 1α,25-dihydroxyvitamin D3. Metabolic activities of CYP1A1/2, CYP2B6, CYP2C9, CYP2C19, CYP3A4/5, UDP-glucuronosyltransferase, and sulfotransferase were also observed in the differentiated cells. In conclusion, MEK, DNMT, and TGF-β inhibitors can be used to promote the differentiation of human iPS cells into pharmacokinetically functional enterocytes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

A gene encoding maize caffeoyl-CoA O-methyltransferase confers quantitative resistance to multiple pathogens.

PubMed

Yang, Qin; He, Yijian; Kabahuma, Mercy; Chaya, Timothy; Kelly, Amy; Borrego, Eli; Bian, Yang; El Kasmi, Farid; Yang, Li; Teixeira, Paulo; Kolkman, Judith; Nelson, Rebecca; Kolomiets, Michael; L Dangl, Jeffery; Wisser, Randall; Caplan, Jeffrey; Li, Xu; Lauter, Nick; Balint-Kurti, Peter

2017-09-01

Alleles that confer multiple disease resistance (MDR) are valuable in crop improvement, although the molecular mechanisms underlying their functions remain largely unknown. A quantitative trait locus, qMdr 9.02 , associated with resistance to three important foliar maize diseases-southern leaf blight, gray leaf spot and northern leaf blight-has been identified on maize chromosome 9. Through fine-mapping, association analysis, expression analysis, insertional mutagenesis and transgenic validation, we demonstrate that ZmCCoAOMT2, which encodes a caffeoyl-CoA O-methyltransferase associated with the phenylpropanoid pathway and lignin production, is the gene within qMdr 9.02 conferring quantitative resistance to both southern leaf blight and gray leaf spot. We suggest that resistance might be caused by allelic variation at the level of both gene expression and amino acid sequence, thus resulting in differences in levels of lignin and other metabolites of the phenylpropanoid pathway and regulation of programmed cell death.

Genetic influences on insight problem solving: the role of catechol-O-methyltransferase (COMT) gene polymorphisms

PubMed Central

Jiang, Weili; Shang, Siyuan; Su, Yanjie

2015-01-01

People may experience an “aha” moment, when suddenly realizing a solution of a puzzling problem. This experience is called insight problem solving. Several findings suggest that catecholamine-related genes may contribute to insight problem solving, among which the catechol-O-methyltransferase (COMT) gene is the most promising candidate. The current study examined 753 healthy individuals to determine the associations between 7 candidate single nucleotide polymorphisms on the COMT gene and insight problem-solving performance, while considering gender differences. The results showed that individuals carrying A allele of rs4680 or T allele of rs4633 scored significantly higher on insight problem-solving tasks, and the COMT gene rs5993883 combined with gender interacted with correct solutions of insight problems, specifically showing that this gene only influenced insight problem-solving performance in males. This study presents the first investigation of the genetic impact on insight problem solving and provides evidence that highlights the role that the COMT gene plays in insight problem solving. PMID:26528222

Genetic influences on insight problem solving: the role of catechol-O-methyltransferase (COMT) gene polymorphisms.

PubMed

Jiang, Weili; Shang, Siyuan; Su, Yanjie

2015-01-01

People may experience an "aha" moment, when suddenly realizing a solution of a puzzling problem. This experience is called insight problem solving. Several findings suggest that catecholamine-related genes may contribute to insight problem solving, among which the catechol-O-methyltransferase (COMT) gene is the most promising candidate. The current study examined 753 healthy individuals to determine the associations between 7 candidate single nucleotide polymorphisms on the COMT gene and insight problem-solving performance, while considering gender differences. The results showed that individuals carrying A allele of rs4680 or T allele of rs4633 scored significantly higher on insight problem-solving tasks, and the COMT gene rs5993883 combined with gender interacted with correct solutions of insight problems, specifically showing that this gene only influenced insight problem-solving performance in males. This study presents the first investigation of the genetic impact on insight problem solving and provides evidence that highlights the role that the COMT gene plays in insight problem solving.

Promoter CpG methylation of multiple genes in pituitary adenomas: frequent involvement of caspase-8.

PubMed

Bello, M Josefa; De Campos, Jose M; Isla, Alberto; Casartelli, Cacilda; Rey, Juan A

2006-02-01

The epigenetic changes in pituitary adenomas were identified by evaluating the methylation status of nine genes (RB1, p14(ARF), p16(INK4a), p73, TIMP-3, MGMT, DAPK, THBS1 and caspase-8) in a series of 35 tumours using methylation-specific PCR analysis plus sequencing. The series included non-functional adenomas (n=23), prolactinomas (n=6), prolactinoma plus thyroid-stimulating hormone adenoma (n=1), growth hormone adenomas (n=4), and adrenocorticotropic adenoma (n=1). All of the tumours had methylation of at least one of these genes and 40% of samples (14 of 35) displayed concurrent methylation of at least three genes. The frequencies of aberrant methylation were: 20% for RB1, 17% for p14(ARF), 34% for p16(INK4a), 29% for p73, 11% for TIMP-3, 23% for MGMT, 6% for DAPK, 43% for THBS1 and 54% for caspase-8. No aberrant methylation was observed in two non-malignant pituitary samples from healthy controls. Although some differences in the frequency of gene methylation between functional and non-functional adenomas were detected, these differences did not reach statistical significance. Our results suggest that promoter methylation is a frequent event in pituitary adenoma tumourigenesis, a process in which inactivation of apoptosis-related genes (DAPK, caspase-8) might play a key role.

Standard (60 Gy) or Short-Course (40 Gy) Irradiation Plus Concomitant and Adjuvant Temozolomide for Elderly Patients With Glioblastoma: A Propensity-Matched Analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minniti, Giuseppe, E-mail: gminniti@ospedalesantandrea.it; Scientific Institute for Research, Hospitalization and Health Care; Scaringi, Claudia

Purpose: To evaluate 2 specific radiation schedules, each combined with temozolomide (TMZ), assessing their efficacy and safety in patients aged ≥65 years with newly diagnosed glioblastoma (GBM). Methods and Materials: Patients aged ≥65 years with Karnofsky performance status (KPS) ≥60 who received either standard (60 Gy) or short-course (40 Gy) radiation therapy (RT) with concomitant and adjuvant TMZ between June 2004 and October 2013 were retrospectively analyzed. A propensity score analysis was executed for a balanced comparison of treatment outcomes. Results: A total of 127 patients received standard RT-TMZ, whereas 116 patients underwent short-course RT-TMZ. Median overall survival and progression-free survival times were similar: 12 monthsmore » and 5.6 months for the standard RT-TMZ group and 12.5 months and 6.7 months for the short-course RT-TMZ group, respectively. Radiation schedule was associated with similar survival outcomes in either unadjusted or adjusted analysis. O{sup 6}-methylguanine-DNA methyltransferase promoter methylation was the most favorable prognostic factor (P=.0001). Standard RT-TMZ therapy was associated with a significant rise in grade 2 and 3 neurologic toxicity (P=.01), lowering of KPS scores during the study (P=.01), and higher posttreatment dosing of corticosteroid (P=.02). Conclusions: In older adults with GBM, survival outcomes of standard and short-course RT-TMZ were similar. An abbreviated course of RT plus TMZ may represent a reasonable therapeutic approach for these patients, without loss of survival benefit and acceptable toxicity.« less

Reserpine Inhibit the JB6 P+ Cell Transformation Through Epigenetic Reactivation of Nrf2-Mediated Anti-oxidative Stress Pathway.

PubMed

Hong, Bo; Su, Zhengyuan; Zhang, Chengyue; Yang, Yuqing; Guo, Yue; Li, Wenjing; Kong, Ah-Ng Tony

2016-05-01

Nuclear factor erythroid-2 related factor 2 (Nrf2) is a crucial transcription factor that regulates the expression of defensive antioxidants and detoxification enzymes in cells. In a previous study, we showed that expression of the Nrf2 gene is regulated by an epigenetic modification. Rauvolfia verticillata, a traditional Chinese herbal medicine widely used in China, possesses anticancer and antioxidant effects. In this study, we investigated how Nrf2 is epigenetically regulated by reserpine, the main active component in R. verticillata, in mouse skin epidermal JB6 P+ cells. Reserpine induced ARE (antioxidant response element)-luciferase activity in HepG2-C8 cells. Accordingly, in JB6 P+ cells, it upregulated the mRNA and protein levels of Nrf2 and its downstream target genes heme oxygenase-1 (HO-1) and quinone oxidoreductase 1 (NQO1), while it only increased the protein level of UDP-glucuronosyltransferase 1A1 (UGT1A1). Furthermore, reserpine decreased the TPA (12-O-tetradecanoylphorbol-13-acetate)-induced colony formation of JB6 cells in a dose-dependent manner. DNA sequencing and methylated DNA immunoprecipitation further demonstrated the demethylation effect of reserpine on the first 15 CpGs of the Nrf2 promoter in JB6 P+ cells. Reserpine also reduced the mRNA and protein expression of DNMT1 (DNA methyltransferase 1), DNMT3a (DNA methyltransferases 3a), and DNMT3b (DNA methyltransferases 3b). Moreover, reserpine induced Nrf2 expression via an epigenetic pathway in skin epidermal JB6 P+ cells, enhancing the protective antioxidant activity and decreasing TPA-induced cell transformation. These results suggest that reserpine exhibits a cancer preventive effect by reactivating Nrf2 and inducing the expression of target genes involved in cellular protection, potentially providing new insight into the chemoprevention of skin cancer using reserpine.

COBALAMIN- AND COBAMIDE-DEPENDENT METHYLTRANSFERASES

PubMed Central

Matthews, Rowena G.; Koutmos, Markos; Datta, Supratim

2008-01-01

Methyltransferases that employ cobalamin cofactors, or their analogues the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple SN2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co+1 intermediates introduces the necessity for complex conformational changes during the catalytic cycle. Recent spectroscopic and structural studies on several of these methyltransferases have helped to reveal the strategies by which these conformational changes are facilitated and controlled. PMID:19059104

Pharmacophore-based virtual screening of catechol-o-methyltransferase (COMT) inhibitors to combat Alzheimer's disease.

PubMed

Patel, Chirag N; Georrge, John J; Modi, Krunal M; Narechania, Moksha B; Patel, Daxesh P; Gonzalez, Frank J; Pandya, Himanshu A

2017-12-27

Alzheimer's disease (AD) is one of the most significant neurodegenerative disorders and its symptoms mostly appear in aged people. Catechol-o-methyltransferase (COMT) is one of the known target enzymes responsible for AD. With the use of 23 known inhibitors of COMT, a query has been generated and validated by screening against the database of 1500 decoys to obtain the GH score and enrichment value. The crucial features of the known inhibitors were evaluated by the online ZINC Pharmer to identify new leads from a ZINC database. Five hundred hits were retrieved from ZINC Pharmer and by ADMET (absorption, distribution, metabolism, excretion, and toxicity) filtering by using FAF-Drug-3 and 36 molecules were considered for molecular docking. From the COMT inhibitors, opicapone, fenoldopam, and quercetin were selected, while ZINC63625100_413 ZINC39411941_412, ZINC63234426_254, ZINC63637968_451, and ZINC64019452_303 were chosen for the molecular dynamics simulation analysis having high binding affinity and structural recognition. This study identified the potential COMT inhibitors through pharmacophore-based inhibitor screening leading to a more complete understanding of molecular-level interactions.

A nonpyrrolysine member of the widely distributed trimethylamine methyltransferase family is a glycine betaine methyltransferase

DOE PAGES

Ticak, Tomislav; Kountz, D. J.; Girosky, K. E.; ...

2014-10-13

COG5598 comprises a large number of proteins related to MttB, the trimethylamine:corrinoid methyltransferase. MttB has a genetically encoded pyrrolysine residue proposed essential for catalysis. MttB is the only known trimethylamine methyltransferase, yet the great majority of members of COG5598 lack pyrrolysine, leaving the activity of these proteins an open question. Here, we describe the function of one of the nonpyrrolysine members of this large protein family. Three nonpyrrolysine MttB homologs are encoded in Desulfitobacterium hafniense, a Gram-positive strict anaerobe present in both the environment and human intestine. D. hafniense was found capable of growth on glycine betaine with electron acceptorsmore » such as nitrate or fumarate, producing dimethylglycine and CO 2 as products. Examination of the genome revealed genes for tetrahydrofolate-linked oxidation of a methyl group originating from a methylated corrinoid protein, but no obvious means to carry out corrinoid methylation with glycine betaine. DSY3156, encoding one of the nonpyrrolysine MttB homologs, was up-regulated during growth on glycine betaine. The recombinant DSY3156 protein converts glycine betaine and cob(I)alamin to dimethylglycine and methylcobalamin. To our knowledge, DSY3156 is the first glycine betaine:corrinoid methyltransferase described, and a designation of MtgB is proposed. Additionally, DSY3157, an adjacently encoded protein, was shown to be a methylcobalamin:tetrahydrofolate methyltransferase and is designated MtgA. Homologs of MtgB are widely distributed, especially in marine bacterioplankton and nitrogen-fixing plant symbionts. Lastly, they are also found in multiple members of the human microbiome, and may play a beneficial role in trimethylamine homeostasis, which in recent years has been directly tied to human cardiovascular health.« less

Structural Chemistry of Human RNA Methyltransferases.

PubMed

Schapira, Matthieu

2016-03-18

RNA methyltransferases (RNMTs) play important roles in RNA stability, splicing, and epigenetic mechanisms. They constitute a promising target class that is underexplored by the medicinal chemistry community. Information of relevance to drug design can be extracted from the rich structural coverage of human RNMTs. In this work, the structural chemistry of this protein family is analyzed in depth. Unlike most methyltransferases, RNMTs generally feature a substrate-binding site that is largely open on the cofactor-binding pocket, favoring the design of bisubstrate inhibitors. Substrate purine or pyrimidines are often sandwiched between hydrophobic walls that can accommodate planar ring systems. When the substrate base is laying on a shallow surface, a 5' flanking base is sometimes anchored in a druggable cavity. The cofactor-binding site is structurally more diverse than in protein methyltransferases and more druggable in SPOUT than in Rossman-fold enzymes. Finally, conformational plasticity observed both at the substrate and cofactor binding sites may be a challenge for structure-based drug design. The landscape drawn here may inform ongoing efforts toward the discovery of the first human RNMT inhibitors.

Expression of hydroxyindole-O-methyltransferase enzyme in the human central nervous system and in pineal parenchymal cell tumors.

PubMed

Fukuda, Takahiro; Akiyama, Nobutake; Ikegami, Masahiro; Takahashi, Hitoshi; Sasaki, Atsushi; Oka, Hidehiro; Komori, Takashi; Tanaka, Yuko; Nakazato, Youichi; Akimoto, Jiro; Tanaka, Masahiko; Okada, Yoshikazu; Saito, Saburo

2010-05-01

Pineal parenchymal tumor (PPT) cells usually show immunoreactivity for synaptophysin, neuron-specific enolase, neurofilament protein, class III beta-tubulin, tau protein, PGP9.5, chromogranin, serotonin, retinal S-antigen, and rhodopsin, but these markers are not specific for PPTs. Melatonin is produced and secreted mainly bypineal parenchymal cells; hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final reaction in melatonin biosynthesis. We hypothesized that HIOMT could serve as a tumor marker of PPTs, and we investigated HIOMT localization and HIOMT expression in samples of normal human tissue and in PPTs, primitive neuroectodermal tumors, and medulloblastomas. In normal tissue, HIOMT was expressed in retinal cells, pineal parenchymal cells, neurons of the Edinger-Westphal nucleus, microglia, macrophages, thyroid follicular epithelium, principal and oxyphil cells of parathyroid gland, adrenal cortical cells, hepatic parenchymal cells, renal tubule epithelium, and enteroendocrine cells of stomach and duodenum. The HIOMT was also expressed in all 46 PPTs studied. The proportions of HIOMT-immunoreactive cells successively decreased in the following tumors: pineocytoma, pineal parenchymal tumor of intermediate differentiation, and pineoblastoma. A few HIOMT-immunoreactive cells were observed in one of 6 primitive neuroectodermal tumors and 23 of 42 medulloblastomas. These results indicate that HIOMT immunohistochemistry may be useful for the diagnosis of PPTs and be a prognostic factor in PPTs.

[Brd3 promotes IL-6 production via enhancing acetylase CBP recruitment and histone 3 acetylation within IL6 promoter].

PubMed

Ren, Wenhui; Sun, Donghao; Wang, Chunmei; Li, Nan

2016-10-01

Objective To investigate the role of bromodomain containing 3 (Brd3) in LPS-triggered interleukin-6 (IL-6) production in macrophages and the underlying mechanism. Methods CRISPR-Cas9 technology was used to screen an RAW264.7 cell line with Brd3 knockout (Brd3 -/- ). The Brd3 -/- cells were used as an experimental group, and the parential cells expressing wide-type Brd3 as a control group. The IL-6 level in cell culture supernatant was detected by ELISA after 100 ng/mL LPS challenging. Effect of Brd3 knockout on the expression and activation of signal pathways involved in IL-6 expression, including the NF-κB and mitogen-activated protein kinase (MAPK) pathways were examined by Western blot analysis. Chromatin immunoprecipitation (ChIP) assay was used to evaluate the recruitment of acetylase CREB-binding protein (CBP) to IL6 gene promoter and the acetylation level of histone 3 within IL6 gene promoter. Results LPS treatment significantly downregulated Brd3 expression in mouse peritoneal macrophages. LPS-induced production of IL-6 was significantly inhibited in Brd3 -/- macrophages. The expressions and activation of signal molecules within NF-κB and MAPK pathways were barely affected. Brd3 knockout significantly decreased the recruitment of acetylase CBP to IL6 gene promoter, and the acetylation level of histone3 within IL6 gene promoter was also repressed. Conclusion Brd3 promotes LPS-triggered IL-6 production via promoting the recruitment of CBP to IL6 promoter and enhancing the acetylation level of histone 3 within IL6 promoter.

Catechol-O-methyltransferase association with hemoglobin A1c

PubMed Central

Hall, Kathryn T.; Jablonski, Kathleen A.; Chen, Ling; Harden, Maegan; Tolkin, Benjamin R.; Kaptchuk, Ted J.; Bray, George A.; Ridker, Paul M.; Florez, Jose C.; Chasman, Daniel I.

2016-01-01

Aims Catecholamines have metabolic effects on blood pressure, insulin sensitivity and blood glucose. Genetic variation in catechol-O-methyltransferase (COMT), an enzyme that degrades catecholamines, is associated with cardiometabolic risk factors and incident cardiovascular disease (CVD). Here we examined COMT effects on glycemic function and type 2 diabetes. Methods We tested whether COMT polymorphisms were associated with baseline HbA1c in the Women’s Genome Health Study (WGHS), and Meta-Analyses of Glucose and Insulin-related traits Consortium (MAGIC), and with susceptibility to type 2 diabetes in WGHS, DIAbetes Genetics Replication And Meta-analysis consortium (DIAGRAM), and the Diabetes Prevention Program (DPP). Given evidence that COMT modifies some drug responses, we examined association with type 2 diabetes and randomized metformin and aspirin treatment. Results COMT rs4680 high-activity G-allele was associated with lower HbA1c in WGHS (β = −0.032% [0.012], p = 0.008) and borderline significant in MAGIC (β = −0.006% [0.003], p = 0.07). Combined COMT per val allele effects on type 2 diabetes were significant (OR = 0.98 [0.96–0.998], p = 0.03) in fixed-effects analyses across WGHS, DIAGRAM, and DPP. Similar results were obtained for 2 other COMT SNPs rs4818 and rs4633. In the DPP, the rs4680 val allele was borderline associated with lower diabetes incidence among participants randomized to metformin (HR = 0.81 [0.65–1.00], p = 0.05). Conclusions COMT rs4680 high-activity G-allele was associated with lower HbA1c and modest protection from type 2 diabetes. The directionality of COMT associations was concordant with those previously observed for cardiometabolic risk factors and CVD. PMID:27282867

FoxO6 regulates memory consolidation and synaptic function

PubMed Central

Salih, Dervis A.M.; Rashid, Asim J.; Colas, Damien; de la Torre-Ubieta, Luis; Zhu, Ruo P.; Morgan, Alexander A.; Santo, Evan E.; Ucar, Duygu; Devarajan, Keerthana; Cole, Christina J.; Madison, Daniel V.; Shamloo, Mehrdad; Butte, Atul J.; Bonni, Azad; Josselyn, Sheena A.; Brunet, Anne

2012-01-01

The FoxO family of transcription factors is known to slow aging downstream from the insulin/IGF (insulin-like growth factor) signaling pathway. The most recently discovered FoxO isoform in mammals, FoxO6, is highly enriched in the adult hippocampus. However, the importance of FoxO factors in cognition is largely unknown. Here we generated mice lacking FoxO6 and found that these mice display normal learning but impaired memory consolidation in contextual fear conditioning and novel object recognition. Using stereotactic injection of viruses into the hippocampus of adult wild-type mice, we found that FoxO6 activity in the adult hippocampus is required for memory consolidation. Genome-wide approaches revealed that FoxO6 regulates a program of genes involved in synaptic function upon learning in the hippocampus. Consistently, FoxO6 deficiency results in decreased dendritic spine density in hippocampal neurons in vitro and in vivo. Thus, FoxO6 may promote memory consolidation by regulating a program coordinating neuronal connectivity in the hippocampus, which could have important implications for physiological and pathological age-dependent decline in memory. PMID:23222102

Chemical Probes of Histone Lysine Methyltransferases

PubMed Central

2015-01-01

Growing evidence suggests that histone methyltransferases (HMTs, also known as protein methyltransferases (PMTs)) play an important role in diverse biological processes and human diseases by regulating gene expression and the chromatin state. Therefore, HMTs have been increasingly recognized by the biomedical community as a class of potential therapeutic targets. High quality chemical probes of HMTs, as tools for deciphering their physiological functions and roles in human diseases and testing therapeutic hypotheses, are critical for advancing this promising field. In this review, we focus on the discovery, characterization, and biological applications of chemical probes for HMTs. PMID:25423077

[A practical guide for the management of gliomas].

PubMed

Stupp, Roger; Pica, Alessia; Mirimanoff, René O; Michielin, Olivier

2007-09-01

The management of gliomas in daily clinical practice is challenging. It requires a multidisciplinary and coordinated approach involving neurosurgery, radiotherapy and, finally, chemotherapy. Important progress has been made during the last years with the introduction of a combined treatment associating standard radiotherapy with concomitant chemotherapy using temozolomide, a novel alkylating agent. For the first time in many years a new treatment strategy translated into a significant prolongation of survival. In parallel, molecular markers (e.g. loss of heterozygosity on chromosomes 1p and 19q or methylation of the methyl-guanine methyl transferase [MGMT] gene promoter) allowed for identification of distinct subtypes of glioma or prediction of treatment response. In this "Practical Guide", we describe the daily practice and aim at answering some common questions in the management of patients suffering from glioblastoma, astrocytoma, oligodendroglioma and low grade glioma. The therapeutic options presented here are based on evidences from the literature. In the absence of documented evidence, the empirical choices from our local practice are explained and justified.

Race moderates the association of Catechol-O-methyltransferase genotype and posttraumatic stress disorder in preschool children.

PubMed

Humphreys, Kathryn L; Scheeringa, Michael S; Drury, Stacy S

2014-10-01

The present study sought to replicate previous findings of an association between the Catechol-O-methyltransferase (COMT) val158met polymorphism with posttraumatic stress disorder (PTSD) and symptomatology in a novel age group, preschool children. COMT genotype was determined in a sample of 171 3-6-year-old trauma-exposed children. PTSD was assessed with a semistructured interview. Accounting for sex, trauma type, and age, genotype was examined in relation to categorical and continuous measures of PTSD both controlling for race and within the two largest racial categories (African American [AA] and European American [EA]). Race significantly moderated the association between genotype and PTSD. Specifically, the genotype associated with increased PTSD symptoms in one racial group had the opposite association in the other racial group. For AA children the met/met genotype was associated with more PTSD symptoms. However, for EA children, val allele carriers had more PTSD symptoms. Whereas every AA child with the met/met genotype met criteria for PTSD, none of the EA children with the met/met genotype did. This genetic association with COMT genotype, in both races but in opposite directions, was most associated with increased arousal symptoms. These findings replicate previous findings in participants of African descent, highlight the moderating effect of race on the association between COMT genotype and PTSD, and provide direct evidence that consideration of population stratification within gene-by-environment studies is valuable to prevent false negative findings.

Race Moderates the Association of Catechol-O-methyltransferase Genotype and Posttraumatic Stress Disorder in Preschool Children

PubMed Central

Humphreys, Kathryn L.; Scheeringa, Michael S.

2014-01-01

Abstract Objective: The present study sought to replicate previous findings of an association between the Catechol-O-methyltransferase (COMT) val158met polymorphism with posttraumatic stress disorder (PTSD) and symptomatology in a novel age group, preschool children. Methods: COMT genotype was determined in a sample of 171 3–6-year-old trauma-exposed children. PTSD was assessed with a semistructured interview. Accounting for sex, trauma type, and age, genotype was examined in relation to categorical and continuous measures of PTSD both controlling for race and within the two largest racial categories (African American [AA] and European American [EA]). Results: Race significantly moderated the association between genotype and PTSD. Specifically, the genotype associated with increased PTSD symptoms in one racial group had the opposite association in the other racial group. For AA children the met/met genotype was associated with more PTSD symptoms. However, for EA children, val allele carriers had more PTSD symptoms. Whereas every AA child with the met/met genotype met criteria for PTSD, none of the EA children with the met/met genotype did. This genetic association with COMT genotype, in both races but in opposite directions, was most associated with increased arousal symptoms. Conclusions: These findings replicate previous findings in participants of African descent, highlight the moderating effect of race on the association between COMT genotype and PTSD, and provide direct evidence that consideration of population stratification within gene-by-environment studies is valuable to prevent false negative findings. PMID:25329975

27 CFR 6.96 - Consumer promotions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2010-04-01 2010-04-01 false Consumer promotions. 6.96 Section 6.96 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS âTIED-HOUSEâ Exceptions § 6.96 Consumer promotions. (a) Coupons. The act by an industry member of furnishing to consumer...

27 CFR 6.96 - Consumer promotions.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 27 Alcohol, Tobacco Products and Firearms 1 2011-04-01 2011-04-01 false Consumer promotions. 6.96 Section 6.96 Alcohol, Tobacco Products and Firearms ALCOHOL AND TOBACCO TAX AND TRADE BUREAU, DEPARTMENT OF THE TREASURY LIQUORS âTIED-HOUSEâ Exceptions § 6.96 Consumer promotions. (a) Coupons. The act by an industry member of furnishing to consumer...

Protein arginine methyltransferase 5 functions as an epigenetic activator of the androgen receptor to promote prostate cancer cell growth.

PubMed

Deng, X; Shao, G; Zhang, H-T; Li, C; Zhang, D; Cheng, L; Elzey, B D; Pili, R; Ratliff, T L; Huang, J; Hu, C-D

2017-03-02

Protein arginine methyltransferase 5 (PRMT5) is an emerging epigenetic enzyme that mainly represses transcription of target genes via symmetric dimethylation of arginine residues on histones H4R3, H3R8 and H2AR3. Accumulating evidence suggests that PRMT5 may function as an oncogene to drive cancer cell growth by epigenetic inactivation of several tumor suppressors. Here, we provide evidence that PRMT5 promotes prostate cancer cell growth by epigenetically activating transcription of the androgen receptor (AR) in prostate cancer cells. Knockdown of PRMT5 or inhibition of PRMT5 by a specific inhibitor reduces the expression of AR and suppresses the growth of multiple AR-positive, but not AR-negative, prostate cancer cells. Significantly, knockdown of PRMT5 in AR-positive LNCaP cells completely suppresses the growth of xenograft tumors in mice. Molecular analysis reveals that PRMT5 binds to the proximal promoter region of the AR gene and contributes mainly to the enriched symmetric dimethylation of H4R3 in the same region. Mechanistically, PRMT5 is recruited to the AR promoter by its interaction with Sp1, the major transcription factor responsible for AR transcription, and forms a complex with Brg1, an ATP-dependent chromatin remodeler, on the proximal promoter region of the AR gene. Furthermore, PRMT5 expression in prostate cancer tissues is significantly higher than that in benign prostatic hyperplasia tissues, and PRMT5 expression correlates positively with AR expression at both the protein and mRNA levels. Taken together, our results identify PRMT5 as a novel epigenetic activator of AR in prostate cancer. Given that inhibiting AR transcriptional activity or androgen synthesis remains the major mechanism of action for most existing anti-androgen agents, our findings also raise an interesting possibility that targeting PRMT5 may represent a novel approach for prostate cancer treatment by eliminating AR expression.

Ada response – a strategy for repair of alkylated DNA in bacteria

PubMed Central

Mielecki, Damian; Grzesiuk, Elżbieta

2014-01-01

Alkylating agents are widespread in the environment and also occur endogenously. They can be cytotoxic or mutagenic to the cells introducing alkylated bases to DNA or RNA. All organisms have evolved multiple DNA repair mechanisms to counteract the effects of DNA alkylation: the most cytotoxic lesion, N3-methyladenine (3meA), is excised by AlkA glycosylase initiating base excision repair (BER); toxic N1-methyladenine (1meA) and N3-methylcytosine (3meC), induced in DNA and RNA, are removed by AlkB dioxygenase; and mutagenic and cytotoxic O6-methylguanine (O6meG) is repaired by Ada methyltransferase. In Escherichia coli, Ada response involves the expression of four genes, ada, alkA, alkB, and aidB, encoding respective proteins Ada, AlkA, AlkB, and AidB. The Ada response is conserved among many bacterial species; however, it can be organized differently, with diverse substrate specificity of the particular proteins. Here, an overview of the organization of the Ada regulon and function of individual proteins is presented. We put special effort into the characterization of AlkB dioxygenases, their substrate specificity, and function in the repair of alkylation lesions in DNA/RNA. PMID:24810496

The Role of the Catechol-o-methyltransferase (COMT) Gene Val158Met in Aggressive Behavior, A Review of Genetic Studies

PubMed Central

Qayyum, Arqam; Zai, Clement C.; Hirata, Yuko; Tiwari, Arun K.; Cheema, Sheraz; Nowrouzi, Behdin; Beitchman, Joseph H.; Kennedy, James L.

2015-01-01

Aggressive behaviors have become a major public health problem, and early-onset aggression can lead to outcomes such as substance abuse, antisocial personality disorder among other issues. In recent years, there has been an increase in research in the molecular and genetic underpinnings of aggressive behavior, and one of the candidate genes codes for the catechol-O-methyltransferase (COMT). COMT is involved in catabolizing catecholamines such as dopamine. These neurotransmitters appear to be involved in regulating mood which can contribute to aggression. The most common gene variant studied in the COMT gene is the Valine (Val) to Methionine (Met) substitution at codon 158. We will be reviewing the current literature on this gene variant in aggressive behavior. PMID:26630958

A Stress-Inducible Resveratrol O-Methyltransferase Involved in the Biosynthesis of Pterostilbene in Grapevine1

PubMed Central

Schmidlin, Laure; Poutaraud, Anne; Claudel, Patricia; Mestre, Pere; Prado, Emilce; Santos-Rosa, Maria; Wiedemann-Merdinoglu, Sabine; Karst, Francis; Merdinoglu, Didier; Hugueney, Philippe

2008-01-01

Stilbenes are considered the most important phytoalexin group in grapevine (Vitis vinifera) and they are known to contribute to the protection against various pathogens. The main stilbenes in grapevine are resveratrol and its derivatives and, among these, pterostilbene has recently attracted much attention due both to its antifungal and pharmacological properties. Indeed, pterostilbene is 5 to 10 times more fungitoxic than resveratrol in vitro and recent studies have shown that pterostilbene exhibits anticancer, hypolipidemic, and antidiabetic properties. A candidate gene approach was used to identify a grapevine resveratrol O-methyltransferase (ROMT) cDNA and the activity of the corresponding protein was characterized after expression in Escherichia coli. Transient coexpression of ROMT and grapevine stilbene synthase in tobacco (Nicotiana benthamiana) using the agroinfiltration technique resulted in the accumulation of pterostilbene in tobacco tissues. Taken together, these results showed that ROMT was able to catalyze the biosynthesis of pterostilbene from resveratrol both in vitro and in planta. ROMT gene expression in grapevine leaves was induced by different stresses, including downy mildew (Plasmopara viticola) infection, ultraviolet light, and AlCl3 treatment. PMID:18799660

Genetic variation in catechol-O-methyltransferase modifies effects of clonidine treatment in chronic fatigue syndrome.

PubMed

Hall, K T; Kossowsky, J; Oberlander, T F; Kaptchuk, T J; Saul, J P; Wyller, V B; Fagermoen, E; Sulheim, D; Gjerstad, J; Winger, A; Mukamal, K J

2016-10-01

Clonidine, an α2-adrenergic receptor agonist, decreases circulating norepinephrine and epinephrine, attenuating sympathetic activity. Although catechol-O-methyltransferase (COMT) metabolizes catecholamines, main effectors of sympathetic function, COMT genetic variation effects on clonidine treatment are unknown. Chronic fatigue syndrome (CFS) is hypothesized to result in part from dysregulated sympathetic function. A candidate gene analysis of COMT rs4680 effects on clinical outcomes in the Norwegian Study of Chronic Fatigue Syndrome in Adolescents: Pathophysiology and Intervention Trial (NorCAPITAL), a randomized double-blinded clonidine versus placebo trial, was conducted (N=104). Patients homozygous for rs4680 high-activity allele randomized to clonidine took 2500 fewer steps compared with placebo (Pinteraction=0.04). There were no differences between clonidine and placebo among patients with COMT low-activity alleles. Similar gene-drug interactions were observed for sleep (Pinteraction=0.003) and quality of life (Pinteraction=0.018). Detrimental effects of clonidine in the subset of CFS patients homozygous for COMT high-activity allele warrant investigation of potential clonidine-COMT interaction effects in other conditions.

Activity of chalcones derived from 2,4,5-trimethoxybenzaldehyde against Meloidogyne exigua and in silico interaction of one chalcone with a putative caffeic acid 3-O-methyltransferase from Meloidogyne incognita.

PubMed

Nunes, Alexandro Silva; Campos, Vicente Paulo; Mascarello, Alessandra; Stumpf, Taisa Regina; Chiaradia-Delatorre, Louise Domenghini; Machado, Alan Rodrigues Teixeira; Santos Júnior, Helvécio Martins; Yunes, Rosendo Augusto; Nunes, Ricardo José; Oliveira, Denilson Ferreira

2013-12-01

Meloidogyne exigua is a parasitic nematode of plants that causes great losses to coffee farmers. In an effort to develop parasitic controls, 154 chalcones were synthesized and screened for activity against this nematode. The best results were obtained with (2E)-1-(4'-nitrophenyl)-3-(2,4,5-trimethoxyphenyl)prop-2-en-1-one (6) with a 50% lethal concentration (LC50) of 171 μg/ml against M. exigua second-stage juveniles, in comparison to the commercially-available nematicide carbofuran which had an LC50 of 260 μg/ml under the same conditions. When coffee plants were used, 6 reduced the nematode population to ~50% of that observed in control plants. To investigate the mechanism of action of 6, an in silico study was carried out, which indicated that 6 may act against M. exigua through inhibition of a putative caffeic acid 3-O-methyltransferase homodimer, the amino acid sequence of which was determined by examining the genome of Meloidogyne incognita. Copyright © 2013 Elsevier Inc. All rights reserved.

Role of Petal-Specific Orcinol O-Methyltransferases in the Evolution of Rose Scent1

PubMed Central

Scalliet, Gabriel; Lionnet, Claire; Le Bechec, Mickaël; Dutron, Laurence; Magnard, Jean-Louis; Baudino, Sylvie; Bergougnoux, Véronique; Jullien, Frédéric; Chambrier, Pierre; Vergne, Philippe; Dumas, Christian; Cock, J. Mark; Hugueney, Philippe

2006-01-01

Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominanty in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells' secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses. PMID:16361520

Enhancing digestibility and ethanol yield of Populus wood via expression of an engineered monolignol 4-O-methyltransferase

PubMed Central

Cai, Yuanheng; Zhang, Kewei; Kim, Hoon; Hou, Guichuan; Zhang, Xuebin; Yang, Huijun; Feng, Huan; Miller, Lisa; Ralph, John; Liu, Chang-Jun

2016-01-01

Producing cellulosic biofuels and bio-based chemicals from woody biomass is impeded by the presence of lignin polymer in the plant cell wall. Manipulating the monolignol biosynthetic pathway offers a promising approach to improved processability, but often impairs plant growth and development. Here, we show that expressing an engineered 4-O-methyltransferase that chemically modifies the phenolic moiety of lignin monomeric precursors, thus preventing their incorporation into the lignin polymer, substantially alters hybrid aspens' lignin content and structure. Woody biomass derived from the transgenic aspens shows a 62% increase in the release of simple sugars and up to a 49% increase in the yield of ethanol when the woody biomass is subjected to enzymatic digestion and yeast-mediated fermentation. Moreover, the cell wall structural changes do not affect growth and biomass production of the trees. Our study provides a useful strategy for tailoring woody biomass for bio-based applications. PMID:27349324

Protein arginine methyltransferase 5 (PRMT5) is a novel coactivator of constitutive androstane receptor (CAR)

DOE Office of Scientific and Technical Information (OSTI.GOV)