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1.
PLoS One ; 15(12): e0242939, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33306687

RESUMO

Transcription factors govern many of the time- and tissue-specific gene expression events in living organisms. CEH-60, a homolog of the TALE transcription factor PBX in vertebrates, was recently characterized as a new regulator of intestinal lipid mobilization in Caenorhabditis elegans. Because CEH-60's orthologs and paralogs exhibit several other functions, notably in neuron and muscle development, and because ceh-60 expression is not limited to the C. elegans intestine, we sought to identify additional functions of CEH-60 through DNA adenine methyltransferase identification (DamID). DamID identifies protein-genome interaction sites through GATC-specific methylation. We here report 872 putative CEH-60 gene targets in young adult animals, and 587 in L2 larvae, many of which are associated with neuron development or muscle structure. In light of this, we investigate morphology and function of ceh-60 expressing AWC neurons, and contraction of pharyngeal muscles. We find no clear functional consequences of loss of ceh-60 in these assays, suggesting that in AWC neurons and pharyngeal muscle, CEH-60 function is likely more subtle or redundant with other factors.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citologia , Caenorhabditis elegans/metabolismo , Metilases de Modificação do DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Músculos/citologia , Neurônios/citologia , Fatores Genéricos de Transcrição/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Ligação Proteica
2.
Ideggyogy Sz ; 73(9-10): 317-325, 2020 Sep 30.
Artigo em Húngaro | MEDLINE | ID: mdl-33035418

RESUMO

Background and purpose: Glioblastoma is the most common malignant CNS tumor, its surgical removal is hindered by the tumors invasive nature, while current anti-tumor therapies show limited effectiveness - mean overall survival is 16-24 months. Some patients show minimal response towards standard oncotherapy, however there are no routinely available prognostic and predictive markers in clinical practice to identify the background of mentioned differences in prognosis. This research aims to identify the prognostic significance of invasion-related extracellular (ECM) components. Methods: Patient groups with different prognoses were created (OS: group A <16 months, group B > 16 months), and internationally recognized prognostic markers (IDH1 mutation and MGMT promoter hyper-methylation) were tested in the flash-frozen tumor samples. Furthermore, the mRNA levels of 46 invasion-related ECM molecules were measured. Results: Clinical data of the patients who have been operated on at the University of Debrecen Clinical Center Department of Neurosurgery and treated at the Department of Clinical Oncology showed no significant differences except for survival data (OS and PFS), and reoperation rate. All samples were IDH wild type. MGMT promoter hypermethylation rate showed significant differences (28.6% vs 68.8%). The expressional pattern of the invasion-related ECM molecules, i.e. the invasion spectrum also showed major differences, integrin ß2, cadherin-12, FLT4/VEGFR-3 and versican molecules having signficantly different mRNA levels. The accuracy of the inivasion spectrum was tested by statistical classifier, 83.3% of the samples was sorted correctly, PPV was 0.93. Conclusion: The difference found in the reoperation rate when comparing different prognostic groups aligns with literature data. MGMG promoter region methylation data in Hungarian samples has not been published yet, and further confirming current knowledge urges the implementation of MGMT promoter analysis in clinical practice. Studying the invasion spectrum provides extra information on tumors, as a prognostic marker it helps recognizing more aggressive tumors, and calls attention to the necessity of using anti-invasive agents in GBM therapies in the future.


Assuntos
Neoplasias Encefálicas/patologia , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Glioblastoma/fisiopatologia , Isocitrato Desidrogenase/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores Tumorais/metabolismo , Glioblastoma/metabolismo , Glioblastoma/cirurgia , Humanos , Prognóstico , RNA Mensageiro
3.
Nat Commun ; 11(1): 3883, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32753598

RESUMO

Temozolomide (TMZ) is an oral alkylating agent used for the treatment of glioblastoma and is now becoming a chemotherapeutic option in patients diagnosed with high-risk low-grade gliomas. The O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for the direct repair of the main TMZ-induced toxic DNA adduct, the O6-Methylguanine lesion. MGMT promoter hypermethylation is currently the only known biomarker for TMZ response in glioblastoma patients. Here we show that a subset of recurrent gliomas carries MGMT genomic rearrangements that lead to MGMT overexpression, independently from changes in its promoter methylation. By leveraging the CRISPR/Cas9 technology we generated some of these MGMT rearrangements in glioma cells and demonstrated that the MGMT genomic rearrangements contribute to TMZ resistance both in vitro and in vivo. Lastly, we showed that such fusions can be detected in tumor-derived exosomes and could potentially represent an early detection marker of tumor recurrence in a subset of patients treated with TMZ.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Rearranjo Gênico , Glioma/tratamento farmacológico , Recidiva Local de Neoplasia/genética , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Adolescente , Adulto , Idoso , Animais , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Adutos de DNA/efeitos dos fármacos , Adutos de DNA/metabolismo , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/prevenção & controle , Regiões Promotoras Genéticas/genética , RNA-Seq , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Sequenciamento Completo do Genoma , Ensaios Antitumorais Modelo de Xenoenxerto , Adulto Jovem
6.
Sci Rep ; 10(1): 9285, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32518380

RESUMO

Most glioblastoma patients have a dismal prognosis, although some survive several years. However, only few biomarkers are available to predict the disease course. EGR1 and EGR3 have been linked to glioblastoma stemness and tumour progression, and this study aimed to investigate their spatial expression and prognostic value in gliomas. Overall 207 gliomas including 190 glioblastomas were EGR1/EGR3 immunostained and quantified. A cohort of 21 glioblastomas with high P53 expression and available tissue from core and periphery was stained with double-immunofluorescence (P53-EGR1 and P53-EGR3) and quantified.EGR1 expression increased with WHO-grade, and declined by 18.9% in the tumour periphery vs. core (P = 0.01), while EGR3 expression increased by 13.8% in the periphery vs. core (P = 0.04). In patients with high EGR1 expression, 83% had methylated MGMT-promoters, while all patients with low EGR1 expression had un-methylated MGMT-promoters. High EGR3 expression in MGMT-methylated patients was associated with poor survival (HR = 1.98; 95%CI 1.22-3.22; P = 0.006), while EGR1 high/EGR3 high, was associated with poor survival vs. EGR1 high/EGR3 low (HR = 2.11; 95%CI 1.25-3.56; P = 0.005). EGR1 did not show prognostic value, but could be involved in MGMT-methylation. Importantly, EGR3 may be implicated in cell migration, while its expression levels seem to be prognostic in MGMT-methylated patients.


Assuntos
Biomarcadores Tumorais/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 3 de Resposta de Crescimento Precoce/genética , Glioblastoma/genética , Glioblastoma/patologia , Movimento Celular/genética , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Progressão da Doença , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Proteína 3 de Resposta de Crescimento Precoce/metabolismo , Feminino , Glioblastoma/diagnóstico , Humanos , Masculino , Prognóstico , Regiões Promotoras Genéticas/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
7.
Tissue Cell ; 62: 101311, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32433023

RESUMO

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. The aim of this study was to investigate the potential effect and mechanism of action of MGMT in strengthing neuronal apoptosis following SCI. To determine MGMT-mediated apoptosis in spinal cord injury, we performed western blot and analyzed the expression change of MGMT with different timepoints. Western blot analysis showed the upregulation of MGMT has a peak at 21 days in injured spinal cord tissues. Expression and location was observed in the neurons after SCI. Upregulation of p53, Bax, cleaved caspase3 and cleaved caspase9 and downregulation of Bcl2 were detected after SCI. Co-localization of cleaved caspase3 with MGMT indicated MGMT involved in apoptosis taking place after SCI. In addition, we carried out H2O2 stimulation to further confirm MGMT played a role in neuron apoptosis process and activated p53 signaling pathway in vitro. Finally, based above data, we packaged lenti-associated virus inhibit MGMT expression and injected into rat spinal cords after SCI model was built. LV-MGMT not only reduces the neuron apoptosis, but also increases GAP43 expression and promotes hindlimbs locomotor function recovery. Taken together, the in vivo data and the in vitro observations prove MGMT-mediated apoptosis in the injured spinal cord.


Assuntos
Apoptose , Metilases de Modificação do DNA/metabolismo , Neurônios/enzimologia , Neurônios/patologia , Traumatismos da Medula Espinal/enzimologia , Traumatismos da Medula Espinal/patologia , Animais , Técnicas de Silenciamento de Genes , Peróxido de Hidrogênio , Injeções , Lentivirus/metabolismo , Masculino , Células PC12 , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia
8.
Brain Tumor Pathol ; 37(2): 50-59, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32361941

RESUMO

Aging is a known negative prognostic factor in glioblastomas (GBM). Whether particular genetic backgrounds are a factor in poor outcomes of elderly patients with GBM warrants investigation. We aim to elucidate any differences between older and younger adult patients with IDH-wildtype GBM regarding both molecular characteristics and clinical outcomes. We collected adult cases diagnosed with IDH-wildtype GBM from the Kansai Network. Clinical and pathological characteristics were analyzed retrospectively and compared between older (≥ 70 years) and younger (≤ 50 years) cases. Included were 92 older vs. 33 younger cases. The older group included more patients with preoperative Karnofsky performance status score < 70 and had a shorter survival time than the younger group. MGMT promoter was methylated more frequently in the older group. TERT promoter mutation was more common in the older group. There were significant differences in DNA copy-number alteration profiles between age groups in PTEN deletion and CDK4 amplification/gain. In the older group, no molecular markers were identified, but surgical resection was an independent prognostic factor. Age-specific survival difference was significant in the MGMT methylated and TERT wildtype subgroup. Elderly patients have several potential factors in poor prognosis of glioblastomas. Varying molecular profiles may explain differing rates of survival between generations.


Assuntos
Neoplasias Encefálicas/genética , Glioblastoma/genética , Isocitrato Desidrogenase/genética , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/mortalidade , Estudos de Coortes , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Feminino , Glioblastoma/mortalidade , Humanos , Japão , Masculino , Metilação , Pessoa de Meia-Idade , Prognóstico , Taxa de Sobrevida , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
9.
Neurochirurgie ; 66(3): 150-154, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32278699

RESUMO

OBJECTIVE: Several parameters are known to predict the survival of glioblastoma (GB), including extent of resection and MGMT promotor methylation. Staining for glial fibrillary acidic protein (GFAP) is a common component of routine histological work-up, but its clinical utility in GB is unclear. The aim of the present study was to analyze the predictive value of quantitative GFAP measurements for survival of patients with GB. METHODS: All subjects in our institutional database of patients with primary GB who underwent surgery between 2011 and 2014 with examination of immunohistochemical staining of GFAP were included. Percentage GFAP staining was measured in 5% increments (5-100%). Univariate and multivariate analyses were performed between GFAP values and survival data. Clinically relevant cut-offs for GFAP staining were identified by receiver operating characteristic (ROC) curves. RESULTS: The final cohort consisted of 272GB patients with available quantitative GFAP measurements (mean age, 62 (±11.1) years, 117 females [43%]). Overall survival was 11.4 months (±8.6). Median GFAP value was 70% (range, 5-100%). The ROC curve showed the clinically relevant cut-off for GFAP at 75% (area under the curve: 0.691). Accordingly, GB patients with GFAP≥75% presented poorer survival on Kaplan-Meier survival estimation (P=0.021). Multivariate analysis adjusted for age, extent of resection, preoperative Karnofsky performance status scale, IDH1 mutation and MGMT methylation status confirmed the independent predictive value of GFAP≥75% for overall survival (P=0.032). Finally, patients with GFAP≥75% showed significantly poorer long-term survival than those with GFAP<75%: 5.8% vs. 15.2% (P=0.0183) and 0.8% vs. 8% (P=0.0076) for 2- and 3-year survival, respectively. CONCLUSION: Quantitative immunohistochemical assessment of GFAP staining could provide a novel biomarker for overall and especially long-term survival of patients with GB. Prospective multi-center validation of the prognostic value of GFAP for GB survival is needed.


Assuntos
Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteína Glial Fibrilar Ácida/biossíntese , Proteína Glial Fibrilar Ácida/genética , Glioblastoma/genética , Glioblastoma/patologia , Idoso , Biomarcadores , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Isocitrato Desidrogenase/biossíntese , Isocitrato Desidrogenase/genética , Estimativa de Kaplan-Meier , Avaliação de Estado de Karnofsky , Masculino , Metilação , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Prognóstico , Análise de Sobrevida , Proteínas Supressoras de Tumor/metabolismo
10.
Nucleic Acids Res ; 48(10): 5397-5406, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32338761

RESUMO

BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction-modification systems, is also active against BREX. In contrast to the wild-type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R-M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.


Assuntos
Bacteriófago T7/fisiologia , Proteínas Virais/metabolismo , Bacteriófago T7/genética , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/virologia , Plasmídeos , Proteínas Virais/genética
11.
PLoS One ; 15(3): e0230492, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32218600

RESUMO

BACKGROUND: Temozolomide (TMZ) has been the standard-of-care chemotherapy for glioblastoma (GBM) patients for more than a decade. Despite this long time in use, significant questions remain regarding how best to optimize TMZ therapy for individual patients. Understanding the relationship between TMZ response and factors such as number of adjuvant TMZ cycles, patient age, patient sex, and image-based tumor features, might help predict which GBM patients would benefit most from TMZ, particularly for those whose tumors lack O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. METHODS AND FINDINGS: Using a cohort of 90 newly-diagnosed GBM patients treated according to the standard of care, we examined the relationships between several patient and tumor characteristics and volumetric and survival outcomes during adjuvant chemotherapy. Volumetric changes in MR imaging abnormalities during adjuvant therapy were used to assess TMZ response. T1Gd volumetric response is associated with younger patient age, increased number of TMZ cycles, longer time to nadir volume, and decreased tumor invasiveness. Moreover, increased adjuvant TMZ cycles corresponded with improved volumetric response only among more nodular tumors, and this volumetric response was associated with improved survival outcomes. Finally, in a subcohort of patients with known MGMT methylation status, methylated tumors were more diffusely invasive than unmethylated tumors, suggesting the improved response in nodular tumors is not driven by a preponderance of MGMT methylated tumors. CONCLUSIONS: Our finding that less diffusely invasive tumors are associated with greater volumetric response to TMZ suggests patients with these tumors may benefit from additional adjuvant TMZ cycles, even for those without MGMT methylation.


Assuntos
Neoplasias Encefálicas , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA de Neoplasias/metabolismo , Glioblastoma , Imagem por Ressonância Magnética , Regiões Promotoras Genéticas , Temozolomida/administração & dosagem , Proteínas Supressoras de Tumor/metabolismo , Adolescente , Adulto , Fatores Etários , Idoso , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Feminino , Glioblastoma/diagnóstico por imagem , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica
12.
Cancer Res ; 80(11): 2101-2113, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32213541

RESUMO

Colorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function. SIGNIFICANCE: This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function.


Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , Metilação de DNA , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Receptores Acoplados a Proteínas-G/biossíntese , Células-Tronco/patologia , Proteína da Polipose Adenomatosa do Colo/deficiência , Proteína da Polipose Adenomatosa do Colo/metabolismo , Animais , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Inativação Gênica , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas-G/genética , Células-Tronco/metabolismo , Via de Sinalização Wnt
13.
Oncogene ; 39(15): 3041-3055, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32066879

RESUMO

The oncogene epidermal growth factor receptor variant III (EGFRvIII) is frequently expressed in glioblastomas (GBM) but its impact on therapy response is still under controversial debate. Here we wanted to test if EGFRvIII influences the sensitivity towards the alkylating agent temozolomide (TMZ). Therefore, we retrospectively analyzed the survival of 336 GBM patients, demonstrating that under standard treatment, which includes TMZ, EGFRvIII expression is associated with prolonged survival, but only in patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated tumors. Using isogenic GBM cell lines with endogenous EGFRvIII expression we could demonstrate that EGFRvIII increases TMZ sensitivity and results in enhanced numbers of DNA double-strand breaks and a pronounced S/G2-phase arrest after TMZ treatment. We observed a higher expression of DNA mismatch repair (MMR) proteins in EGFRvIII+ cells and patient tumor samples, which was most pronounced for MSH2 and MSH6. EGFRvIII-specific knockdown reduced MMR protein expression thereby increasing TMZ resistance. Subsequent functional kinome profiling revealed an increased activation of p38- and ERK1/2-dependent signaling in EGFRvIII expressing cells, which regulates MMR protein expression downstream of EGFRvIII. In summary, our results demonstrate that the oncoprotein EGFRvIII sensitizes a fraction of GBM to current standard of care treatment through the upregulation of DNA MMR.


Assuntos
Neoplasias Encefálicas/terapia , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Glioblastoma/terapia , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Quimiorradioterapia/métodos , Estudos de Coortes , Metilação de DNA , Reparo de Erro de Pareamento de DNA/efeitos dos fármacos , Reparo de Erro de Pareamento de DNA/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/mortalidade , Humanos , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Proteína 2 Homóloga a MutS/genética , Mutação , Regiões Promotoras Genéticas/genética , Estudos Retrospectivos , Temozolomida/uso terapêutico , Proteínas Supressoras de Tumor/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
14.
Int J Mol Sci ; 21(3)2020 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-32023925

RESUMO

DNA methylation is a process through which methyl groups are added to the DNA molecule, thereby modifying the activity of a DNA segment without changing the sequence. Increasing evidence has shown that DNA methylation is involved in various aspects of plant growth and development via a number of key processes including genomic imprinting and repression of transposable elements. DNA methylase and demethylase are two crucial enzymes that play significant roles in dynamically maintaining genome DNA methylation status in plants. In this work, 22 DNA methylase genes and six DNA demethylase genes were identified in rapeseed (Brassica napus L.) genome. These DNA methylase and DNA demethylase genes can be classified into four (BnaCMTs, BnaMET1s, BnaDRMs and BnaDNMT2s) and three (BnaDMEs, BnaDML3s and BnaROS1s) subfamilies, respectively. Further analysis of gene structure and conserved domains showed that each sub-class is highly conserved between rapeseed and Arabidopsis. Expression analysis conducted by RNA-seq as well as qRT-PCR suggested that these DNA methylation/demethylation-related genes may be involved in the heat/salt stress responses in rapeseed. Taken together, our findings may provide valuable information for future functional characterization of these two types of epigenetic regulatory enzymes in polyploid species such as rapeseed, as well as for analyzing their evolutionary relationships within the plant kingdom.


Assuntos
Brassica napus/crescimento & desenvolvimento , Metilação de DNA , Perfilação da Expressão Gênica/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brassica napus/genética , Brassica napus/metabolismo , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Epigênese Genética , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico , Família Multigênica , Filogenia , Proteínas de Plantas/química , Domínios Proteicos , Estresse Salino , Análise de Sequência de RNA , Distribuição Tecidual
15.
Biomed Pharmacother ; 125: 109896, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32007918

RESUMO

Cisplatin (DDP) is the first-line drug for the treatment of gastric cancer (GC). However, DDP resistance is common. Autophagy, which is closely related to chemoresistance, is a process of resolving and recycling proteins and damaged cellular organs. Additionally, O-6-methylguanine-DNA methyltransferase (MGMT) is responsible for alkylating drug resistance. However, the relationship between autophagy and MGMT in response to DDP in GC is still unknown. In the present study, we determined that autophagy induced by DDP decreases chemosensitivity in GC cell lines. DDP may have induced autophagy in GC by inhibiting MGMT to increase autophagy-related gene (ATG) 4B. Inhibition of MGMT-mediated ATG4B suppression resulted in autophagy induction and DDP resistance. In vivo, combined DDP and autophagy inhibitor chloroquine (CQ) enhanced the anti-tumor effect of DDP; additionally, the negative correlation of MGMT and ATG4B was confirmed. High expression of MGMT and low expression of ATG4B were significantly correlated with favorable five-year survival rate (P < 0.05) in 66 clinicopathologically characterized GC cases. Our study demonstrate that DDP inhibits MGMT-mediated autophagy suppression to decrease chemosensitivity in GC, which provides a novel therapeutic strategy to promote DDP chemosensitivity in GC.


Assuntos
Antineoplásicos/uso terapêutico , Autofagia/efeitos dos fármacos , Cisplatino/uso terapêutico , Metilases de Modificação do DNA/antagonistas & inibidores , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/antagonistas & inibidores , Enzimas Reparadoras do DNA/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos/farmacologia , Autofagia/fisiologia , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Distribuição Aleatória , Neoplasias Gástricas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
16.
Toxicology ; 435: 152413, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32109525

RESUMO

DNA interstrand cross-links (ICLs) are essential for the antitumor activity of chloroethylnitrosoureas (CENUs). Commonly, CENUs resistance is mainly considered to be associated with O6-methylguanine-DNA methyltransferase (MGMT) within tumors. Bypassing the MGMT-mediated resistance, to our knowledge, herein, we first utilized a novel glycolytic inhibitor, 3-bromopyruvate (3-BrPA), to increase the cytotoxic effects of l,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) to human glioma cells based on the hypothesis that blocking energy metabolism renders tumor cells more sensitive to chemotherapy. We found 3-BrPA significantly increased the cell killing by BCNU in human glioma SF763 and SF126 cell lines. Significantly decreased levels of extracellular lactate, cellular ATP and glutathione (GSH) were observed after 3-BrPA treatment, and the effects were more remarkable with 3-BrPA in combination with BCNU. Considering that the role of ATP and GSH in drug efflux, DNA damage repair and drug inactivation, we determined the effect of 3-BrPA on the formation of dG-dC ICLs induced by BCNU using stable isotope dilution high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS). As expected, the levels of lethal dG-dC ICLs induced by BCNU were obviously enhanced after 3-BrPA pretreatment. Based on these results, 3-BrPA and related glycolytic inhibitors may be promising to enhance the cell killing effect and reverse the clinical chemoresistance of CENUs and related antitumor agents.


Assuntos
Antineoplásicos Alquilantes/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Carmustina/farmacologia , Dano ao DNA , Glioma/tratamento farmacológico , Glicólise/efeitos dos fármacos , Piruvatos/farmacologia , Trifosfato de Adenosina/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Resistência a Medicamentos , Glioma/metabolismo , Glioma/patologia , Glutationa/metabolismo , Humanos , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Proteínas Supressoras de Tumor/metabolismo
17.
J Clin Neurosci ; 73: 231-236, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32070670

RESUMO

Although promoter methylation status is known to correlate with response to alkylating agents, immunohistochemistry (IHC) is the only available method for MGMT status in many institutions. However, the clinical utility of MGMT IHC is controversial. A hundred and twenty four cases of primary glioblastoma diagnosed by morphology-based criteria over 2 decades were re-appraised based on WHO 2016 classification. Tumor MGMT status was evaluated by IHC and methylation-specific PCR (MSP) to see if any correlation between the results of the 2 methods. The association with patients' prognoses was also investigated. Among 124 cases, 116 were confirmed to be glioblastoma by definition of WHO2016, and median overall survival (OS) of glioblastoma, IDH-wildtype and epithelioid glioblastoma were 18 and 27 months, respectively. MGMT promoter methylation status significantly correlated with progression-free survival (PFS) (p = 0.014) but not with OS (p = 0.364) in patients with glioblastoma by the integrated diagnosis. With the cut-off value of 24.5% of positive cell ratio as determined by receiver operating characteristic (ROC) analysis, there was a good correlation between the results of IHC and MSP (p = 0.08 × 10-24). Accordingly, there was a marginal association between the results of IHC and patients' PFS (p = 0.063), but not OS (p = 0.563). When the patients were divided into pre and post temozolomide era, the association of MGMT promoter methylation status with PFS was only noted in the patients of temozolomide era (pre, p = 0.432; post, p = 0.015).


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Encefálicas/diagnóstico , Metilação de DNA , Metilases de Modificação do DNA/genética , Glioblastoma/patologia , Adulto , Idoso , Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/genética , Metilases de Modificação do DNA/metabolismo , Feminino , Glioblastoma/genética , Humanos , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Regiões Promotoras Genéticas , Curva ROC
18.
Int Immunopharmacol ; 80: 106147, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31931367

RESUMO

DNA methylation, especially DNA methyltransferases (DNMTs), is involved in the pathogenesis of many autoimmune diseases through regulating immune function. This study aimed to explore the potential role of DNMTs in IgA nephropathy (IgAN). We evaluated mRNA expressions of DNMT1, DNMT3A, DNMT3B along with ß1,3-galactosyltransferase (C1GALT1) in peripheral blood mononuclear cells (PBMCs), and measured galactose-deficient IgA1 (Gd-IgA1) levels in plasma. The expression intensity of DNMT1 and DNMT3B in the renal specimen of IgAN patients were also detected. Results showed DNMT3B, not DNMT1 or DNMT3A, was notably increased in IgAN patients compared to controls and associated with pathologic types. However, DNMT1 and C1GALT1 were found positively correlated with estimated glomerular filtration rate (eGFR) and negatively correlated with 24 h Urine protein in IgAN patients. No association was found between DNMT1 and Gd-IgA1. The expressions of DNMT3B and DNMT1 were barely observed in IgAN renal biopsy specimens. In conclusion, for the first time, we identified the relations of DNMTs and C1GALT1 to the clinical state and pathology of IgAN patients, which provide new clues for IgAN.


Assuntos
Metilases de Modificação do DNA/genética , Galactosiltransferases/genética , Glomerulonefrite por IGA/enzimologia , Adulto , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Feminino , Glomerulonefrite por IGA/sangue , Glomerulonefrite por IGA/patologia , Humanos , Imunoglobulina A/sangue , Rim/enzimologia , Rim/patologia , Leucócitos Mononucleares/metabolismo , Masculino , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Adulto Jovem
19.
Talanta ; 210: 120609, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-31987176

RESUMO

DNA methylation is involved in the oncogenesis of head and neck squamous cell carcinoma and could be used for early detection of cancer to increase the chances of cure, but unfortunately diagnosis is usually made at late stages of the disease. In this work we developed genosensors to detect DNA methylation of the MGMT gene in head and neck cancer cell lines. The probe for MGMT promoter methylation was immobilized on gold electrodes modified with 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayers (SAM). Detection was performed with electrochemical impedance spectroscopy, with clear distinction between methylated and non-methylated DNA from head and neck cell lines. The genosensor is sensitive with a low detection limit of 0.24 × 10-12 mol L-1. In addition, the cell lines FaDu, JHU28 and SCC25 for the MGMT gene, could be distinguished from the HN13 cell line which has a high degree of MGMT methylation (97%), thus confirming the selectivity. Samples with different percentages of MGMT DNA methylation could be separated in multidimensional projections using the visualization technique interactive document mapping (IDMAP). The genosensor matrix and the immobilization procedures are generic, and can be extended to other DNA methylation biomarkers.


Assuntos
Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Técnicas Eletroquímicas , Ácidos Graxos/química , Neoplasias de Cabeça e Pescoço/genética , Compostos de Sulfidrila/química , Tioglicolatos/química , Proteínas Supressoras de Tumor/genética , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Eletrodos , Ouro/química , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Metilação , Regiões Promotoras Genéticas/genética , Espectrofotometria Infravermelho , Proteínas Supressoras de Tumor/metabolismo
20.
FASEB J ; 34(1): 1038-1051, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914596

RESUMO

Over recent years several examples of randomly switching methyltransferases, associated with Type III restriction-modification (R-M) systems, have been described in pathogenic bacteria. In every case examined, changes in simple DNA sequence repeats result in variable methyltransferase expression and result in global changes in gene expression, and differentiation of the bacterial cell into distinct phenotypes. These epigenetic regulatory systems are called phasevarions, phase-variable regulons, and are widespread in bacteria, with 17.4% of Type III R-M system containing simple DNA sequence repeats. A distinct, recombination-driven random switching system has also been described in Streptococci in Type I R-M systems that also regulate gene expression. Here, we interrogate the most extensive and well-curated database of R-M systems, REBASE, by searching for all possible simple DNA sequence repeats in the hsdRMS genes that encode Type I R-M systems. We report that 7.9% of hsdS, 2% of hsdM, and of 4.3% of hsdR genes contain simple sequence repeats that are capable of mediating phase variation. Phase variation of both hsdM and hsdS genes will lead to differential methyltransferase expression or specificity, and thereby the potential to control phasevarions. These data suggest that in addition to well characterized phasevarions controlled by Type III mod genes, and the previously described Streptococcal Type I R-M systems that switch via recombination, approximately 10% of all Type I R-M systems surveyed herein have independently evolved the ability to randomly switch expression via simple DNA sequence repeats.


Assuntos
Epigênese Genética , Repetições de Microssatélites , Regulon , Proteínas de Bactérias/genética , Biologia Computacional , DNA/análise , Metilação de DNA , Metilases de Modificação do DNA/metabolismo , Enzimas de Restrição-Modificação do DNA/genética , Enzimas de Restrição-Modificação do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo I/genética , Fusobacterium nucleatum , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Mannheimia haemolytica , Metiltransferases/metabolismo , Fenótipo , Pseudomonas aeruginosa , Salmonella enterica
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