Pulsed and Discontinuous Electromagnetic Field Exposure Decreases Temozolomide Resistance in Glioblastoma by Modulating the Expression of O6-Methylguanine-DNA Methyltransferase, Cyclin-D1, and p53.

Background: Glioblastoma is a malignant and very aggressive brain tumor with a poor prognosis. Despite having chemotherapy concomitant with surgery and/or radiation therapy, the median survival of glioblastoma-affected people is less than 1 year. Temozolomide (TMZ) is a chemotherapeutic used as a first line treatment of glioblastoma. Several studies have reported that resistance to TMZ due to overexpression of O6-methylguanine-DNA methyltransferase (MGMT) is the main reason for treatment failure. Several studies described that pulsed-electromagnetic field (EMF) exposure could induce cell death and influence gene expression. Materials and Methods: In this study the authors assessed the effects of EMF (50 Hz, 70 G) on cytotoxicity, cell migration, gene expression, and protein levels in TMZ-treated T98 and A172 cell lines. Results: In this study, the authors show that treatment with a combination of TMZ and EMF enhanced cell death and decreased the migration potential of T98 and A172 cells. The authors also observed overexpression of the p53 gene and downregulation of cyclin-D1 protein in comparison to controls. In addition, T98 cells expressed the MGMT protein following treatment, while the A172 cells did not express MGMT. Conclusion: Their data indicate that EMF exposure improved the cytotoxicity of TMZ on T98 and A172 cells and could partially affect resistance to TMZ in T98 cells.

PLEKHG5 regulates autophagy, survival and MGMT expression in U251-MG glioblastoma cells.

A signalling pathway involving PLEKHG5 (guanine exchange factor) for the Ras superfamily member RAB26 to transcription factor NF-κB was discovered in autophagy. PLEKHG5 was reported in glioblastoma multiforme (GBM) and correlates with patient survival. Thus, the generation of a cellular model for understanding PLEKHG5 signalling is the study purpose. We generated a CRISPR/Cas9-mediated knockout of PLEKHG5 in U251-MG glioblastoma cells and analysed resulting changes. Next, we used a mRFP-GFP-LC3+ reporter for visualisation of autophagic defects and rescued the phenotype of PLEKHG5 wildtype via transduction of a constitutively active RAB26QL-plasmid. Effects of overexpressing RAB26 were investigated and correlated with the O6-methylguanine-DNA methyltransferase (MGMT) and cellular survival. PLEKHG5 knockout showed changes in morphology, loss of filopodia and higher population doubling times. Accumulation of autolysosomes was resulted by decreased LAMP-1 in PLEKHG5-deficient cells. Rescue of PLEKHG5-/- restored the downregulation of RhoA activity, showed faster response to tumour necrosis factor and better cellular fitness. MGMT expression was activated after RAB26 overexpression compared to non-transduced cells. Survival of PLEKHG5 knockout was rescued together with sensitivity to temozolomide by RAB26QL. This study provides new insights in the PLEKHG5/RAB26 signalling within U251-MG cells, which suggests potential therapeutic strategies in other glioma cells and further in primary GBM.

Cedrol, a Sesquiterpene Alcohol, Enhances the Anticancer Efficacy of Temozolomide in Attenuating Drug Resistance via Regulation of the DNA Damage Response and MGMT Expression.

Glioblastoma (GBM) is a common and aggressive brain tumor with a median survival of 12-15 months. Temozolomide (TMZ) is a first-line chemotherapeutic agent used in GBM therapy, but the occurrence of drug resistance limits its antitumor activity. The natural compound cedrol has remarkable antitumor activity and is derived from Cedrus atlantica. In this study, we investigated the combined effect of TMZ and cedrol in GBM cells in vitro and in vivo. The TMZ and cedrol combination treatment resulted in consistently higher suppression of cell proliferation via regulation of the AKT and MAPK signaling pathways in GBM cells. The combination treatment induced cell cycle arrest, cell apoptosis, and DNA damage better than either drug alone. Furthermore, cedrol reduced the expression of proteins associated with drug resistance, including O6-methlyguanine-DNA-methyltransferase (MGMT), multidrug resistance protein 1 (MDR1), and CD133 in TMZ-treated GBM cells. In the animal study, the combination treatment significantly suppressed tumor growth through the induction of cell apoptosis and decreased TMZ drug resistance. Moreover, cedrol-treated mice exhibited no significant differences in body weight and improved TMZ-induced liver damage. These results imply that cedrol may be a potential novel agent for combination treatment with TMZ for GBM therapy that deserves further investigation.

MGMT expression affects the gemcitabine resistance of pancreatic cancer cells.

Pancreatic cancer is a malignant cancer with poor prognosis. This study aimed to explore how O6-methylguanine-DNA methyltransferase (MGMT) affects the gemcitabine resistance of pancreatic cancer cells by the regulatory role of SHH/GLI signaling pathway. MGMT inhibition induced by lomeguatrib (LM) suppressed the proliferation, invasion, migration and autophagy, promoted the apoptosis of PanC-1/GEM cells and up-regulated the GEM inhibition rates for PanC-1/GEM cells. Moreover, MGMT inhibition increased the expression of Caspase-3 and Bax and decreased the expression of Bcl-2, Beclin1 and Atg5 in PanC-1/GEM cells. PVT1 silencing could also produce the similar effects of MGMT inhibition induced by LM on PanC-1/GEM cells. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in PanC-1/GEM cells by regulating the MGMT expression. miR-409 was demonstrated to be a potential target of PVT1 and SHH was demonstrated to be a potential target of miR-409. Furthermore, GLI overexpression could reverse the effects of PVT1 silencing. In the xenograft model of pancreatic cancer, nude mice were treated with GEM. MGMT inhibition suppressed the tumor growth and autophagy and promoted the apoptosis in tumor tissues. And, PVT1 silencing could inhibit the SHH/GLI signaling pathway in tumor tissues. In conclusion, MGMT inhibition could suppress the proliferation, invasion, migration and autophagy and promote the apoptosis of PanC-1/GEM cells in vitro and in vivo. PVT1 silencing may affect the PanC-1/GEM cells through changing the MGMT expression by inhibiting the SHH/GLI signaling pathway.

Expression and prognosis analysis of DNMT family in acute myeloid leukemia.

DNA methyltransferases (DNMTs) by regulating DNA methylation play crucial roles in the progression of hematologic malignancies, especially for acute myeloid leukemia (AML). Accumulating investigations have identified the high incidence of DNMT3A mutation in AML, and it is correlated with poor prognosis. Although a few studies have shown the expression of DNMTs and their clinical significance in AML, the results remain to be discussed. Herein, we systemically analyzed the DNMTs expression and their relationship with clinic-pathological features and prognosis in AML patients. DNMTs expression especially for DNMT3A/3B was closely associated with AML among various human cancers. DNMT3A expression was increased in AML patients, whereas DNMT3B expression was decreased. Significant associations between DNMT3A/B expression and clinic-pathological features/gene mutations were observed. Kaplan-Meier analysis showed that DNMT3A expression was associated with better overall survival (OS) and leukemia-free survival (LFS) among whole-cohort AML, and independently affected OS determined by Cox repression multivariate analysis. Notably, patients that received hematopoietic stem cell transplantation (HSCT) showed significantly better OS and LFS in DNMT3A lower-expressed groups, whereas patients in DNMT3A higher-expressed groups did not. By bioinformatics analysis, DNMT3A expression was found to be positively correlated with several leukemia-associated genes/microRNAs, and DNMT3A was identified as direct targets of miR-429 and miR-29b in AML. Collectively, our study demonstrated that DNMT3A/3B showed significant expression differences in AML. DNMT3A expression acted as a potential prognostic biomarker and may guide treatment choice between chemotherapy and HSCT in AML.

Granulocyte-macrophage colony-stimulating factor enhances effect of temozolomide on high-grade glioma cells.

In the present study, to delve into the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with temozolomide (TMZ) on high-grade glioma cells and related mechanism, six cases of high-grade glioma cells from patient's tumor tissues were cultured. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed to detect cell proliferation and toxicity. Flow cytometry was performed to ascertain cell cycle and apoptosis rate. To detect the expressions of O6-methylguanine-DNA methyltransferase (MGMT) methylation status and MGMT protein, respectively, specific PCR and immunofluorescence were performed. According to the results of MTT assay, compared with the results of control group, GM-CSF group exhibited enhanced cell viability in varying degrees. In three cases of cells (MGMT gene methylation), the combination group [(67.67 ± 1.16), (68.13 ± 1.06), (68.42 ± 1.73)] had noticeably lower cell viability than the corresponding TMZ group [(90.00 ± 1.73), (82.33 ± 1.53), (82.67 ± 2.11)] (P < 0.01). Nevertheless, the two groups showed no significant difference in another three cases (MGMT gene unmethylated) (P > 0.05). In combination group, the apoptosis rate of the MGMT methylation cells was higher than that in the corresponding TMZ group (P < 0.01), which is consistent with MTT assay results. In all six cases of primary glioma cells, the fraction of cells in G1 phase of GM-CSF-treated group was noticeably down-regulated and was up-regulated in S phase (P < 0.01). GM-CSF could induce high-grade glioma cells to rapidly enter the cell cycle, thereby enhancing the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation. However, this effect is not ideal on glioma cells with MGMT unmethylation.

Riluzole enhances the antitumor effects of temozolomide via suppression of MGMT expression in glioblastoma.

OBJECTIVE: Glutamatergic signaling significantly promotes proliferation, migration, and invasion in glioblastoma (GBM). Riluzole, a metabotropic glutamate receptor 1 inhibitor, reportedly suppresses GBM growth. However, the effects of combining riluzole with the primary GBM chemotherapeutic agent, temozolomide (TMZ), are unknown. This study aimed to investigate the efficacy of combinatorial therapy with TMZ/riluzole for GBM in vitro and in vivo. METHODS: Three GBM cell lines, T98G (human; O6-methylguanine DNA methyltransferase [MGMT] positive), U87MG (human; MGMT negative), and GL261 (murine; MGMT positive), were treated with TMZ, riluzole, or a combination of both. The authors performed cell viability assays, followed by isobologram analysis, to evaluate the effects of combinatorial treatment for each GBM cell line. They tested the effect of riluzole on MGMT, a DNA repair enzyme causing chemoresistance to TMZ, through quantitative real-time reverse transcription polymerase chain reaction in T98G cells. Furthermore, they evaluated the efficacy of combinatorial TMZ/riluzole treatment in an orthotopic mouse allograft model of MGMT-positive GBM using C57BL/6 J mice and GL261 cells. RESULTS: Riluzole displayed significant time- and dose-dependent growth-inhibitory effects on all GBM cell lines assessed independently. Riluzole enhanced the antitumor effect of TMZ synergistically in MGMT-positive but not in MGMT-negative GBM cell lines. Riluzole singularly suppressed MGMT expression, and it significantly suppressed TMZ-induced MGMT upregulation (p < 0.01). Furthermore, combinatorial TMZ/riluzole treatment significantly suppressed tumor growth in the intracranial MGMT-positive GBM model (p < 0.05). CONCLUSIONS: Riluzole attenuates TMZ-induced MGMT upregulation and enhances the antitumor effect of TMZ in MGMT-positive GBMs. Therefore, combinatorial TMZ/riluzole treatment is a potentially promising novel therapeutic regimen for MGMT-positive GBMs.

MGMT expression in pituitary corticotroph adenomas and its relationship to clinical, pathological, and ultrastructural parameters in patients with Cushing's disease.

INTRODUCTION: Transsphenoidal surgery is the treatment of choice in Cushing's disease (CD), although even late recurrences occur in some patients. Low expression of O-6-methylguanine-DNA methyltransferase (MGMT) has been linked to a high risk of relapse in pituitary tumours, but the evidence for corticotroph adenomas is limited. Therefore, we investigated whether MGMT expression was associated with CD remission or clinicopathological markers of tumour aggressiveness among patients with corticotroph adenomas. MATERIAL AND METHODS: We included 72 consecutive patients (83% female, mean age ±SD: 44.15 ±15.15 years) with CD, who underwent transsphenoidal adenomectomy between 2012 and 2018. The invasiveness of corticotroph tumours was assessed based on the Knosp scale. Immunohistochemistry was used to analyse MGMT expression as well as the proliferation markers (Ki-67, p53, mitotic index). Electron microscopy was used to categorise tumours into densely or sparsely granulated. Early biochemical remission was evaluated in all patients 6 months after pituitary surgery. RESULTS: Early remission was observed in 47 (65%) patients 6 months after surgery. MGMT expression was > 75% in half of all tumours, < 25% in 14 tumours, and 25-50% or 50-75% in 11 tumours. Lower MGMT expression was associated with a larger tumour diameter (p = 0.001), higher adrenocorticotropic hormone (ACTH) concentration (p = 0.002), higher p53 expression (p = 0.026), and higher frequency of sparsely granulated corticotroph adenomas (p = 0.009). Low MGMT expression was significantly related to lower frequency of early clinical remission (p = 0.005). CONCLUSIONS: MGMT predicted the outcomes of transsphenoidal surgery for CD. Pituitary corticotroph adenomas with low MGMT expression may be associated with increased invasiveness and poorer prognosis.

Exposure to ultrafine particulate matter induces NF-κß mediated epigenetic modifications.

Exposure to ultrafine particulate matter (PM0.1) is positively associated with the etiology of different acute and chronic disorders; however, the in-depth biological imprints that link these submicron particles with the disturbances in the epigenomic machinery are not well defined. Earlier, we showed that exposure to these particles causes significant disturbances in the mitochondrial machinery and triggers PI-3-kinase mediated DNA damage responses. In the present study, we aimed to further understand the epigenomic insights of the ultrafine PM exposure. The higher levels of intracellular reactive oxygen species and depleted Nrf-2 in ultrafine PM exposed cells reconfirmed its potential to induce oxidative stress. Importantly, the observed increase in the levels of NF-κß and associated cytokines among exposed cells suggested the activation of NF-κß mediated inflammatory loop which potentially serves as a platform for initiating epigenetic insinuations. This fact was strongly supported by the altered miRNA expression profile of the ultrafine PM exposed cells. These NF-κß induced miRNA alterations were also found to be associated with other epigenetic targets as the exposed cells showed higher expression levels of DNA methyltransferases which positively corresponded with the global changes in DNA methylation levels. Upon further analysis, significant alterations in histone code were also reported in ultrafine PM exposed cells. Conclusively our results suggested that NF-κß acts as an inflammatory switch that possesses the potential to induce genome-wide epigenetic modification upon ultrafine PM exposure.

Epigenetic modulation of BRCA-1 and MGMT genes, and histones H4 and H3 are associated with breast tumors.

Aberrant patterns in promoter methylation of tumor-suppressor genes and posttranslational modifications of histone proteins are considered as major features of malignancy. In this study, we aimed to investigate promoter methylation of three tumor-suppressor genes (BRCA-1, MGMT, and P16) and three histone marks (H3K9ac, H3K18ac, and H4K20me3) in patients with breast tumors. This case-control study included 27 patients with malignant breast tumors (MBT) and 31 patients with benign breast tumors (BBT). The methylation-specific PCR was used for determining promoter methylation of BRCA-1, MGMT, and P16 genes. Western blot analysis was performed to detect histone lysine acetylation (H3K9ac and H3K18ac) and lysine methylation (H4K20me3). BRCA-1 promoter methylation was detected in 44.4% of the MBT whereas this alteration was found in 9.7% of BBT (P = 0.005). The Kaplan-Meier analysis indicated that hypermethylation in BRCA-1 promoter was significantly associated with poor overall survival of patients with breast cancer (P = 0.039). MGMT promoter methylation was identified in 18.5% of MBT and 0.0% of the BBT (P = 0.01). The frequency of P16 promoter methylation was 25.8% in BBT and 11.1% in MBT (P = 0.12). As compared with BBT, MBT samples displayed the aberrant patterns of histones marks with hypomethylation of H4K20 and hypoacetylation of H3K18 (P = 0.03 and P = 0.04, respectively). There was a negative significant correlation between H3K9ac levels and tumor size in MBT group (r = -0.672; P = 0.008). The present findings suggest that promoter hypermethylation of MGMT and BRCA-1 genes along with alterations in H3K18ac and H4K20me3 levels may have prognostic values in patients with breast cancer. Moreover, the detection of these epigenetic modifications in breast tumors could be helpful in finding new methods for breast cancer therapy.

MIR29B mediates epigenetic mechanisms of HBG gene activation.

Sickle cell disease (SCD) affects over 2 million people worldwide with high morbidity and mortality in underdeveloped countries. Therapeutic interventions aimed at reactivating fetal haemoglobin (HbF) is an effective approach for improving survival and ameliorating the clinical severity of SCD. A class of agents that inhibit DNA methyltransferase (DNMT) activity show promise as HbF inducers because off-target effects are not observed at low concentrations. However, these compounds are rapidly degraded by cytidine deaminase when taken by oral administration, creating a critical barrier to clinical development for SCD. We previously demonstrated that microRNA29B (MIR29B) inhibits de novo DNMT synthesis, therefore, the goal of our study was to determine if MIR29 mediates HbF induction. Overexpression of MIR29B in human KU812 cells and primary erythroid progenitors significantly increased the percentage of HbF positive cells, while decreasing the expression of DNMT3A and the HBG repressor MYB. Furthermore, HBG promoter methylation levels decreased significantly following MIR29B overexpression in human erythroid progenitors. We subsequently, observed higher MIR29B expression in SCD patients with higher HbF levels compared to those with low HbF. Our findings provide evidence for the ability of MIR29B to induce HbF and supports further investigation to expand treatment options for SCD.

Diffusion Kurtosis Imaging Reflects Glial Fibrillary Acidic Protein (GFAP), Topo IIα, and O6-Methylguanine-DNA Methyltransferase (MGMT) Expression in Astrocytomas.

BACKGROUND Astrocytomas are the most common primary brain neoplasms. Biological indicators of astrocytomas can reflect its biological characteristics. The aim of this study was to assess the expression of the pathological glial fibrillary acidic protein (GFAP) Topo IIα and O6-methylguanine-DNA methyltransferase (MGMT) in astrocytomas using magnetic resonance (MR) diffusion kurtosis imaging (DKI) to evaluate the biological characteristics of astrocytomas. MATERIAL AND METHODS Sixty-six patients with pathologically proven astrocytomas were enrolled in this study. All patients underwent conventional MRI head scanning, DKI scanning, and enhanced scanning under the same conditions. Spearman's rank correlation analysis and Bonferroni correction were used to compare the values of DKI and the expression levels of GFAP, Topo IIα, and MGMT between the 2 groups. RESULTS Mean kurtosis (MK) values were negatively correlated with the expression of GFAP (r=-0.836; P=0.03). However, these were positively correlated with the expression of Topo IIα (r=0.896; P=0.01). Moreover, fractional anisotropy (FA) values were not correlated with the expression of GFAP (r=0.366; P=0.05), Topo IIα (r=-0.562; P=0.05), or MGMT (r=-0.153; P=0.10). CONCLUSIONS MK was significantly associated with the expression of GFAP and Topo IIα. To a certain extent, applying DKI may show the biological behavior of tumor cell differentiation, proliferation activity, invasion, and metastasis, and guide individual treatment.

Predicting O6-Methylguanine-DNA Methyltransferase Protein Expression in Primary Low- and High-Grade Gliomas Using Certain Qualitative Characteristics of Amide Proton Transfer-Weighted Magnetic Resonance Imaging.

OBJECTIVE: To demonstrate that certain qualitative amide proton transfer-weighted (APTw) characteristics can provide practical imaging clues for predicting O6-methylguanine-DNA methyltransferase (MGMT) protein expression in primary low- and high-grade gliomas, preoperatively and noninvasively. METHODS: Pathologically confirmed low- and high-grade gliomas with APT data and immunohistochemical (IHC) reports were recruited in this study. The MGMT protein expression status was classified by postsurgery specimen immunostaining. Subjects were divided into two groups, MGMT-positive and MGMT-negative group, according to the immunoreactivity of MGMT protein expression documented in IHC reports. APTw images scanned at 3T magnetic resonance preoperatively were retrospectively analyzed. Two neuroradiologists were trained to evaluate presence of certain APTw features. Kappa value was calculated to show the consistency between the 2 observers. The Mann-Whitney U test was used to evaluate relationships between the 2 groups on APTw features. Negative predictive value and positive predictive value was used to evaluate the ability of APTw characteristics in predicting MGMT protein expression. Receiver operating characteristic curve was used to evaluate the diagnostic performance of APTw characters. Two-tailed P < 0.05 was considered as statistically significant. RESULTS: Forty-two subjects were recruited in this study. Among them 38 specimens presented positive MGMT immunostaining (MGMT-positive group), 4 specimens were negative MGMT immunostaing (MGMT-negative group). There were, respectively, 37 and 5 APTw images appeared positive and negative APTw features. Differences between tumors of positive and negative MGMT expression on qualitative APTw features were significant (P = 0.020). The consistency coefficient of the 2 observers was 0.876 (kappa = 0.876). Three of five llgliomas with negative APTw features showed MGMT-negative immunostaining, leading to a negative predictive value of 60%, and 36 of 37 cases presenting positive APTw characteristics were tumors of MGMT-positive expression, generating a positive predictive value of 97.3%. The area under curve was 0.849. CONCLUSIONS: APTw characteristics could be promising imaging markers by which to predict IHC MGMT expression in primary low- and high-grade gliomas preoperatively and noninvasively.

Proteins of the retinoblastoma pathway, FEN1 and MGMT are novel potential prognostic biomarkers in pancreatic adenocarcinoma.

BACKGROUND: We studied the expression of some major proteins involved in cell-cycle regulation and DNA repair, the roles of which are not well known in pancreatic ductal adenocarcinoma (PDAC), but which have a significant impact on carcinogenesis of many other cancers. METHODS: We immunohistochemically assessed expression levels of the cell-cycle regulators Rb1, p16 and cyclin-dependent kinase 4 (CDK4), and the DNA repair enzymes O6-methylguanine-DNA-alkyltransferase (MGMT) and flap endonuclease-1 (FEN1) separately in malignant tissue and benign tissue from resection margins in 102 cases of PDAC. Nearly all (95.1%) patients had undergone pancreaticoduodenectomy. RESULTS: The studied proteins showed wide but somewhat variable expression in both benign and malignant pancreatic tissues. Strong CDK4 expression in islets of Langerhans predicted poor relapse-free survival (RFS) (HR 2.874; 95% CI 1.261-6.550; p = .012) and within T3-4 tumors CDK4 expression in adenocarcinoma cells also predicted poor disease-free survival (DFS) (RR 2.148; 95% CI 1.081-4.272; p = .029). Strong MGMT expression was associated in N1 patients with weak local relapse-free survival (RFS), DFS and overall survival; all significantly in Cox regression analysis. FEN1 was also an independent predictor of decreased DFS (in the whole study population) and worse RFS (in the patients with T3-4 tumors). CONCLUSIONS: Major cell-cycle regulator also have predictive significance, but further studies are required to evaluate this.

Functional analyses of PtRDM1 gene overexpression in poplars and evaluation of its effect on DNA methylation and response to salt stress.

Epigenetic modification by DNA methylation is necessary for all cellular processes, including genetic expression events, DNA repair, genomic imprinting and regulation of tissue development. It occurs almost exclusively at the C5 position of symmetric CpG and asymmetric CpHpG and CpHpH sites in genomic DNA. The RNA-directed DNA methylation (RDM1) gene is crucial for heterochromatin and DNA methylation. We overexpressed PtRDM1 gene from Populus trichocarpa to amplify transcripts of orthologous RDM1 in 'Nanlin895' (P. deltoides × P. euramericana 'Nanlin895'). This overexpression resulted in increasing RDM1 transcript levels: by ∼150% at 0 mM NaCl treatment and by ∼300% at 60 mM NaCl treatment compared to WT (control) poplars. Genomic cytosine methylation was monitored within 5.8S rDNA and histone H3 loci by bisulfite sequencing. In total, transgenic poplars revealed more DNA methylation than WT plants. In our results, roots revealed more methylated CG contexts than stems and leaves whereas, histone H3 presented more DNA methylation than 5.8S rDNA in both WT and transgenic poplars. The NaCl stresses enhanced more DNA methylation in transgenic poplars than WT plants through histone H3 and 5.8 rDNA loci. Also, the overexpression of PtRDM1 resulted in hyper-methylation, which affected plant phenotype. Transgenic poplars revealed significantly more regeneration of roots than WT poplars via NaCl treatments. Our results proved that RDM1 protein enhanced the DNA methylation by chromatin remodeling (e.g. histone H3) more than repetitive DNA sequences (e.g. 5.8S rDNA).

The impact of polyunsaturated fatty acids on DNA methylation and expression of DNMTs in human colorectal cancer cells.

Growing evidence suggests a role of polyunsaturated fatty acids (PUFA) in the prevention of various types of malignancy, including colorectal cancer (CRC). No published studies have yet examined the direct effect of PUFA treatment on DNA methylation in CRC cells. In this study, 5 human CRC cells were treated with 100 µM DHA, EPA, and LA for 6 days and changes in their global- and gene-specific DNA methylation status as well as expression of DNA methyl transferases (DNMT) were investigated. Cell-type specific differences in DNA methylation and expression of DNMTs were observed in PUFA-treated cells. DHA and EPA treatment induced global hypermethylation in HT29/219 and HCT116 cells, but reduced methylation in Caco2 cells (p < 0.05). Among 10 tumor related genes tested in 5 CRC cell lines, DHA and EPA induced promoter demethylation of Cox2 in HT29/219, p14 and PPARγ in HCT116, and ECAD in SW742 cells. Cell-type specific differences in expression of DNMT1, DNMT3a, and 3b genes were also observed between PUFA-treated and control cells (p < 0.05). Overall, treatment of PUFAs coordinately induced the expression of DNMTs in HT29/219, but suppressed in other 4 cell lines investigated in this study.

Enhancing the cytotoxicity of chemoradiation with radiation-guided delivery of anti-MGMT morpholino oligonucleotides in non-methylated solid tumors.

The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) is epigenetically silenced in some tumors by MGMT gene promoter methylation. MGMT-hypermethylated solid tumors have enhanced susceptibility to the cytotoxic effects of alkylating chemotherapy such as temozolomide, compared with non-methylated tumors. In glioblastoma, subjects with MGMT hypermethylation have significantly longer survival rates after chemoradiotherapy. We report the first successful use of a non-ablative dose of ionizing radiation to prime human cancer cells to enhance the uptake of unmodified anti-MGMT morpholino oligonucleotide (AMON) sequences. We demonstrate >40% reduction in the in vitro proliferation index and cell viability in radiation-primed MGMT-expressing human solid tumor cells treated with a single dose of AMONs and temozolomide. We further demonstrate the feasibility of using a non-ablative dose of radiation in vivo to guide and enhance the delivery of intravenously administered AMONs to achieve 50% MGMT knockdown only at radiation-primed tumor sites in a subcutaneous tumor model. Local upregulation of physiological endocytosis after radiation may have a role in radiation-guided uptake of AMONs. This approach holds direct translational significance in glioblastoma and brain metastases where radiation is part of the standard of care; our approach to silence MGMT could overcome the significant problem of MGMT-mediated chemoresistance.

Decreased expression of MGMT in correlation with aberrant DNA methylation in esophageal cancer patients from North India.

O-6-methylguanine-DNA methyltransferase, DNA repair gene, has been found to be involved with the pathogenesis of the esophageal cancer. DNA hypermethylation and other factors have been suggested to downregulate O-6-methylguanine-DNA methyltransferase. In this communication, the methylation status of O-6-methylguanine-DNA methyltransferase gene and the corresponding O-6-methylguanine-DNA methyltransferase protein expression in esophageal cancer from North India has been studied. In all, 80 samples of tumor tissue along with adjacent normal tissue as controls were analyzed for messenger RNA level of O-6-methylguanine-DNA methyltransferase gene, protein expression, and subcellular localization. The messenger RNA expression was studied using real-time quantitative polymerase chain reaction, protein expression, and its subcellular localization by Western blotting and immunohistochemistry. DNA methylation was assessed through methylation-specific polymerase chain reaction. Clinicopathological parameters were recorded and correlated with the O-6-methylguanine-DNA methyltransferase expression. O-6-methylguanine-DNA methyltransferase messenger RNA expression was found to be downregulated in 65% cases (52/80). The expression of O-6-methylguanine-DNA methyltransferase at the protein level was also found to be absent in 65% (52/80) cases. In all, 52 cases had low or no expression of the protein, whereas out of those 28 remaining cases, 11.25% (09/80) cases had high O-6-methylguanine-DNA methyltransferase protein expression. The absence of O-6-methylguanine-DNA methyltransferase protein coincided with the methylated cases in 84% (38/45), whereas in 07 cases, out of the 45 methylated, O-6-methylguanine-DNA methyltransferase protein was present. The aggressive esophageal cancer patients having methylated O-6-methylguanine-DNA methyltransferase had more than 50% cases with no/mild expression of the O-6-methylguanine-DNA methyltransferase protein ( p > 0.001). Loss of O-6-methylguanine-DNA methyltransferase protein was very frequent in the incidence of esophageal cancer from North Indian patients, and methylation of the promoter region of O-6-methylguanine-DNA methyltransferase was significantly associated in its downregulation.

Alteration in the expression of MGMT and RUNX3 due to non-CpG promoter methylation and their correlation with different risk factors in esophageal cancer patients.

Promoter methylation reflects in the inactivation of different genes like O6-methylguanine-DNA methyltransferase DNA repair gene and runt-related transcription factor 3, a known tumor suppressor gene in various cancers such as esophageal cancer. The promoter methylation was evaluated for O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in CpG, CHH, and CHG context (where H is A, T, or C) by next-generation sequencing. The methylation status was correlated with quantitative messenger RNA expression. In addition, messenger RNA expression was correlated with different risk factors like tobacco, alcohol, betel nut consumption, and smoking habit. CpG methylation of O6-methylguanine-DNA methyltransferase promoter had a positive association in the development of esophageal cancer (p < 0.05), whereas runt-related transcription factor 3 promoter methylation showed no significant association (p = 1.0) to develop esophageal cancer. However, the non-CpG methylation, CHH, and CHG were significantly correlated with O6-methylguanine-DNA methyltransferase (p < 0.05) and runt-related transcription factor 3 (p < 0.05) promoters in the development of esophageal cancer. The number of cytosine converted to thymine (C→T) in O6-methylguanine-DNA methyltransferase promoter showed a significant correlation between cases and controls (p < 0.05), but in runt-related transcription factor 3 no such significant correlation was observed. Besides, messenger RNA expression was found to be significantly correlated with promoter hypermethylation of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 in the context of CHG and CHH (p < 0.05). The CpG hypermethylation in O6-methylguanine-DNA methyltransferase showed positive (p < 0.05) association, whereas in runt-related transcription factor 3, it showed contrasting negative association (p = 0.23) with their messenger RNA expression. Tobacco, betel nut consumption, and smoking habits were associated with altered messenger RNA expression of O6-methylguanine-DNA methyltransferase (p < 0.05) and betel nut consumption and smoking habits were associated with runt-related transcription factor 3 (p < 0.05). There was no significant association between messenger RNA expression of O6-methylguanine-DNA methyltransferase and runt-related transcription factor 3 with alcohol consumption (p = 0.32 and p = 0.15). In conclusion, our results suggest that an aberrant messenger RNA expression may be the outcome of CpG, CHG, and CHH methylation in O6-methylguanine-DNA methyltransferase, whereas outcome of CHG and CHH methylation in runt-related transcription factor 3 promoters along with risk factors such as consumption of tobacco, betel nut, and smoking habits in esophageal cancer from Northeast India.

MGMT promoter methylation status in Merkel cell carcinoma: in vitro versus invivo.

PURPOSE: Expression of O6-methylguanine-DNA methyltransferase (MGMT) in Merkel cell carcinoma (MCC) is very variable; thus, we tested whether this may be due to differential methylation of the MGMT gene promoter. METHODS: Quantitative analysis of MGMT mRNA and protein expression, as well as MGMT promoter methylation status, was performed in a series of tissue samples of MCC tumors, representing both primary and metastatic lesions, as well as in six MCC cell lines. RESULTS: These analyses revealed a very heterogeneous MGMT mRNA and protein expression in MCC both in vivo and in vitro. However, neither the MGMT mRNA nor protein expression correlated with the sensitivity of MCC cell lines toward the alkylating agent dacarbazine in vitro. Notably, increased methylation at the promoter of the MGMT gene was observed in 2/6 (33%) of the MCC cell lines; however, MGMT promoter methylation was absent in all MCC tissue samples. According to our results, albeit aberrant methylation of MGMT gene promoter can be observed in in vitro propagated MCC cell lines, it seems to be absent or very rare in MCC lesions in situ. CONCLUSION: Thus, the evaluation of this marker has no or only little significance for predicting response to therapy or for improving efficacy of demethylating agents in the treatment of MCC. Microenvironmental factors may play a role in explaining the different results between MCC cell lines and MCC samples.