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1.
Int J Mol Sci ; 19(8)2018 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065170

RESUMO

The survival kinase protein kinase B (Akt) participates in the regulation of essential subcellular processes, e.g., proliferation, growth, survival, and apoptosis, and has a documented role in promoting resistance against genotoxic stress including radiotherapy, presumably by influencing the DNA damage response and DNA double-strand break (DSB) repair. However, its exact role in DSB repair requires further elucidation. We used a genetic approach to explore the consequences of impaired phosphorylation of Akt1 at one or both of its key phosphorylation sites, Threonine 308 (T308) or Serine 473 (S473), on DSB repair and radiosensitivity to killing. Therefore, we overexpressed either the respective single or the double phosphorylation-deficient mutants (Akt1-T308A, Akt1-S473A, or Akt1-T308A/S473A) in TRAMPC1 murine prostate cancer cells (TrC1) and measured the DSB repair kinetics and clonogenic cell survival upon irradiation. Only the expression of the Akt1-T308A/S473A induced a significant delay in the kinetics of DSB repair in irradiated TrC1 as determined by the γH2A.X (H2A histone family, member X) assay and the neutral comet assay, respectively. Moreover, Akt1-T308A/S473A-expressing cells were characterized by increased radiosensitivity compared to Akt1-WT (wild type)-expressing cells in long-term colony formation assays. Our data reveal that Akt1's activation state is important for the cellular radiation response, presumably by modulating the phosphorylation of effector proteins involved in the regulation of DSB repair.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos da radiação , Fosforilação/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos da radiação , Ensaio Cometa , Reparo do DNA/efeitos da radiação , Camundongos
2.
Int J Mol Sci ; 19(12)2018 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-30486506

RESUMO

Proton beam therapy is increasingly applied for the treatment of human cancer, as it promises to reduce normal tissue damage. However, little is known about the relationship between linear energy transfer (LET), the type of DNA damage, and cellular repair mechanisms, particularly for cells irradiated with protons. We irradiated cultured cells delivering equal doses of X-ray photons, Bragg-peak protons, or plateau protons and used this set-up to quantitate initial DNA damage (mainly DNA double strand breaks (DSBs)), and to analyze kinetics of repair by detecting γH2A.X or 53BP1 using immunofluorescence. The results obtained validate the reliability of our set-up in delivering equal radiation doses under all conditions employed. Although the initial numbers of γH2A.X and 53BP1 foci scored were similar under the different irradiation conditions, it was notable that the maximum foci level was reached at 60 min after irradiation with Bragg-peak protons, as compared to 30 min for plateau protons and photons. Interestingly, Bragg-peak protons induced larger and irregularly shaped γH2A.X and 53BP1 foci. Additionally, the resolution of these foci was delayed. These results suggest that Bragg-peak protons induce DNA damage of increased complexity which is difficult to process by the cellular repair apparatus.


Assuntos
Reparo do DNA/efeitos da radiação , Transferência Linear de Energia/efeitos da radiação , Fótons , Raios X , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Imunofluorescência , Transferência Linear de Energia/genética
3.
Postepy Hig Med Dosw (Online) ; 69: 140-52, 2015 Jan 23.
Artigo em Polonês | MEDLINE | ID: mdl-25614681

RESUMO

The transcription factor Nrf2 controls the expression of genes encoding cytoprotective enzymes and proteins. Its activation is related to conformational changes in the inhibitory protein Keap1 and/or Nrf2 phosphorylation by upstream kinases. Activation of Nrf2 can lead to the induction of phase II enzymes responsible for the inactivation of potential carcinogens. This may constitute an important strategy of chemoprevention. Moreover, these enzymatic systems participating in the biotransformation of drugs can reduce their therapeutic effects, contributing to drug resistance. For this reason, a clear understanding of the role of Nrf2 is essential to assess the beneficial and adverse effects of its up-regulation, particularly in relation to the prevention and treatment of cancer. This article summarizes the current state of knowledge on the significance of Nrf2 in tumorigenesis.


Assuntos
Citoproteção/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Animais , Carcinogênese , Regulação da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteína 1 Associada a ECH Semelhante a Kelch , Redes e Vias Metabólicas , Fator 2 Relacionado a NF-E2/química , Fosforilação , Relação Estrutura-Atividade
4.
Oncotarget ; 15: 699-713, 2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39352803

RESUMO

Monoclonal antibody therapies for cancer have demonstrated extraordinary clinical success in recent years. However, these strategies are thus far mostly limited to specific cell surface antigens, even though many disease targets are found intracellularly. Here we report studies on the humanization of a full-length, nucleic acid binding, monoclonal lupus-derived autoantibody, 3E10, which exhibits a novel mechanism of cell penetration and tumor specific targeting. Comparing humanized variants of 3E10, we demonstrate that cell uptake depends on the nucleoside transporter ENT2, and that faster cell uptake and superior in vivo tumor targeting are associated with higher affinity nucleic acid binding. We show that one human variant retains the ability of the parental 3E10 to bind RAD51, serving as a synthetically lethal inhibitor of homology-directed repair in vitro. These results provide the basis for the rational design of a novel antibody platform for therapeutic tumor targeting with high specificity following systemic administration.


Assuntos
Rad51 Recombinase , Humanos , Animais , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Rad51 Recombinase/imunologia , Camundongos , Linhagem Celular Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Peptídeos Penetradores de Células/farmacologia , Peptídeos Penetradores de Células/química
5.
Front Genet ; 13: 884210, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35711920

RESUMO

The BRCA2 germline missense variant, R3052W, resides in the DNA binding domain and has been previously classified as a pathogenic allele. In this study, we sought to determine how R3052W alters the cellular functions of BRCA2 in the DNA damage response. The BRCA2 R3052W mutated protein exacerbates genome instability, is unable to rescue homology-directed repair, and fails to complement cell survival following exposure to PARP inhibitors and crosslinking drugs. Surprisingly, despite anticipated defects in DNA binding or RAD51-mediated DNA strand exchange, the BRCA2 R3052W protein mislocalizes to the cytoplasm precluding its ability to perform any DNA repair functions. Rather than acting as a simple loss-of-function mutation, R3052W behaves as a dominant negative allele, likely by sequestering RAD51 in the cytoplasm.

6.
Nat Biotechnol ; 40(3): 325-334, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34711990

RESUMO

Gene amplification drives oncogenesis in a broad spectrum of cancers. A number of drugs have been developed to inhibit the protein products of amplified driver genes, but their clinical efficacy is often hampered by drug resistance. Here, we introduce a therapeutic strategy for targeting cancer-associated gene amplifications by activating the DNA damage response with triplex-forming oligonucleotides (TFOs), which drive the induction of apoptosis in tumors, whereas cells without amplifications process lower levels of DNA damage. Focusing on cancers driven by HER2 amplification, we find that TFOs targeting HER2 induce copy number-dependent DNA double-strand breaks (DSBs) and activate p53-independent apoptosis in HER2-positive cancer cells and human tumor xenografts via a mechanism that is independent of HER2 cellular function. This strategy has demonstrated in vivo efficacy comparable to that of current precision medicines and provided a feasible alternative to combat drug resistance in HER2-positive breast and ovarian cancer models. These findings offer a general strategy for targeting tumors with amplified genomic loci.


Assuntos
Neoplasias da Mama , Amplificação de Genes , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Dano ao DNA , Feminino , Genômica , Humanos , Oligonucleotídeos
7.
Mol Cancer Res ; 19(12): 2057-2067, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34535560

RESUMO

Exploitation of DNA repair defects has enabled major advances in treating specific cancers. Recent work discovered that the oncometabolite 2-hydroxyglutarate (2-HG), produced by neomorphic isocitrate dehydrogenase 1/2 (IDH1/2) mutations, confers a homology-directed repair (HDR) defect through 2-HG-induced histone hypermethylation masking HDR signaling. Here, we report that IDH1-mutant cancer cells are profoundly sensitive to the histone deacetylase inhibitor (HDACi) vorinostat, by further suppressing the residual HDR in 2-HG-producing cells. Vorinostat downregulates repair factors BRCA1 and RAD51 via disrupted E2F-factor regulation, causing increased DNA double-strand breaks, reduced DNA repair factor foci, and functional HDR deficiency even beyond 2-HG's effects. This results in greater cell death of IDH1-mutant cells and confers synergy with radiation and PARPi, both against cells in culture and patient-derived tumor xenografts. Our work identifies HDACi's utility against IDH1-mutant cancers, and presents IDH1/2 mutations as potential biomarkers to guide trials testing HDACi in gliomas and other malignancies. IMPLICATIONS: IDH1-mutant cells show profound vulnerability to HDACi treatment, alone and with PARPi and radiation, via HDR suppression, presenting IDH1/2 mutations as biomarkers for HDACi use in gliomas and other malignancies.


Assuntos
Reparo do DNA/genética , Glioma/tratamento farmacológico , Inibidores de Histona Desacetilases/uso terapêutico , Isocitrato Desidrogenase/metabolismo , Animais , Linhagem Celular Tumoral , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Camundongos Nus
8.
iScience ; 24(11): 103366, 2021 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-34825138

RESUMO

Cancer bioenergetics fuel processes necessary to maintain viability and growth under stress conditions. We hypothesized that cancer metabolism supports the repair of radiation-induced DNA double-stranded breaks (DSBs). We combined the systematic collection of metabolic and radiobiological data from a panel of irradiated cancer cell lines with mathematical modeling and identified a common metabolic response with impact on the DSB repair kinetics, including a mitochondrial shutdown followed by compensatory glycolysis and resumption of mitochondrial function. Combining ionizing radiation (IR) with inhibitors of the compensatory glycolysis or mitochondrial respiratory chain slowed mitochondrial recovery and DNA repair kinetics, offering an opportunity for therapeutic intervention. Mathematical modeling allowed us to generate new hypotheses on general and individual mechanisms of the radiation response with relevance to DNA repair and on metabolic vulnerabilities induced by cancer radiotherapy. These discoveries will guide future mechanistic studies for the discovery of metabolic targets for overcoming intrinsic or therapy-induced radioresistance.

9.
Cells ; 9(4)2020 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-32260562

RESUMO

Technical improvements in clinical radiotherapy for maximizing cytotoxicity to the tumor while limiting negative impact on co-irradiated healthy tissues include the increasing use of particle therapy (e.g., proton therapy) worldwide. Yet potential differences in the biology of DNA damage induction and repair between irradiation with X-ray photons and protons remain elusive. We compared the differences in DNA double strand break (DSB) repair and survival of cells compromised in non-homologous end joining (NHEJ), homologous recombination repair (HRR) or both, after irradiation with an equal dose of X-ray photons, entrance plateau (EP) protons, and mid spread-out Bragg peak (SOBP) protons. We used super-resolution microscopy to investigate potential differences in spatial distribution of DNA damage foci upon irradiation. While DNA damage foci were equally distributed throughout the nucleus after X-ray photon irradiation, we observed more clustered DNA damage foci upon proton irradiation. Furthermore, deficiency in essential NHEJ proteins delayed DNA repair kinetics and sensitized cells to both, X-ray photon and proton irradiation, whereas deficiency in HRR proteins sensitized cells only to proton irradiation. We assume that NHEJ is indispensable for processing DNA DSB independent of the irradiation source, whereas the importance of HRR rises with increasing energy of applied irradiation.


Assuntos
Reparo do DNA por Junção de Extremidades/efeitos da radiação , Prótons , Reparo de DNA por Recombinação/efeitos da radiação , Animais , Linhagem Celular , Sobrevivência Celular/efeitos da radiação , Células Clonais , Dano ao DNA , DNA Ligase Dependente de ATP/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Histonas/metabolismo , Humanos , Camundongos , Fótons , Fatores de Tempo , Raios X
10.
Sci Rep ; 9(1): 3148, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30816253

RESUMO

DNA- and histone-related research frequently comprises the quantitative analysis of protein modifications, such as histone phosphorylation. Analysis of accumulation and disappearance of protein foci are used to monitor DNA damage and repair kinetics. If the protein of interest doesn't accumulate in foci, laser micro-irradiation of single nuclei provides an alternative method to monitor DNA repair proteins and histone dynamics at the DNA damage site. We have developed an automated evaluation tool for standardized, high-throughput analysis of micro-irradiated cells featuring single cell background subtraction and detection across multiple fluorescence channels, allowing for robust statistics.


Assuntos
Código das Histonas/genética , Histonas/genética , Processamento de Proteína Pós-Traducional/genética , Proteínas/genética , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/genética , Dano ao DNA/efeitos da radiação , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Histonas/química , Humanos , Lasers/efeitos adversos , Microscopia de Fluorescência , Fosforilação/efeitos da radiação , Proteínas/química , Análise de Célula Única
11.
Radiat Res ; 188(1): 114-120, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28492345

RESUMO

The quantitative analysis of foci plays an important role in various cell biological methods. In the fields of radiation biology and experimental oncology, the effect of ionizing radiation, chemotherapy or molecularly targeted drugs on DNA damage induction and repair is frequently performed by the analysis of protein clusters or phosphorylated proteins recruited to so called repair foci at DNA damage sites, involving for example γ-H2A.X, 53BP1 or RAD51. We recently developed "The Focinator" as a reliable and fast tool for automated quantitative and qualitative analysis of nuclei and DNA damage foci. The refined software is now even more user-friendly due to a graphical interface and further features. Thus, we included an R-script-based mode for automated image opening, file naming, progress monitoring and an error report. Consequently, the evaluation no longer required the attendance of the operator after initial parameter definition. Moreover, the Focinator v2-0 is now able to perform multi-channel analysis of four channels and evaluation of protein-protein colocalization by comparison of up to three foci channels. This enables for example the quantification of foci in cells of a specific cell cycle phase.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos da radiação , Gráficos por Computador , Dano ao DNA/genética , Microscopia de Fluorescência/métodos , Software , Interface Usuário-Computador , Células A549 , Algoritmos , Núcleo Celular/efeitos da radiação , Núcleo Celular/ultraestrutura , Citometria de Fluxo/métodos , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Linguagens de Programação
12.
Oncotarget ; 6(18): 16663-73, 2015 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-26143636

RESUMO

Telomerase reverse transcriptase (TERT) activity is up-regulated in several types of tumors including glioblastoma (GBM). In the present study, 128 primary glioblastoma patients were examined for single nucleotide polymorphisms of TERT in blood and in 92 cases for TERT promoter mutations in tumors. TERT promoter mutations were observed in 86% of the tumors and of these, C228T (-124 bp upstream start codon) was detected in 75% and C250T (-146 bp) in 25% of cases. TERT promoter mutations were associated with shorter overall survival (11 vs. 20 months p = 0.002 and 12 vs. 20, p = 0.04 for C228T and C250T, respectively). The minor alleles of rs2736100 and rs10069690 SNP's, located in intron 2 and the promotor regions, respectively, were associated with an increased risk of developing GBM (p = 0.004 and 0.001). GBM patients having both TERT promoter mutations and being homozygous carriers of the rs2853669 C-allele displayed significantly shorter overall survival than those with the wild type allele. The rs2853669 SNP is located in a putative Ets2 binding site in the promoter (-246 bp upstream start codon) close to the C228T and C250T mutation hot spots. Interleukin-6 (IL-6) expression regulated by TERT promoter status and polymorphism, what leads us to think that TERT and IL-6 plays a significant role in GBM, where specific SNPs increase the risk of developing GBM while the rs2853669 SNP and specific mutations in the TERT promoter of the tumor lead to shorter survival.


Assuntos
Biomarcadores Tumorais/genética , Glioblastoma/genética , Regiões Promotoras Genéticas/genética , Telomerase/genética , Sítios de Ligação/genética , Feminino , Glioblastoma/mortalidade , Glioblastoma/patologia , Humanos , Interleucina-1beta/genética , Interleucina-6/genética , Isocitrato Desidrogenase/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Prognóstico , RNA Mensageiro/biossíntese , Complexo Shelterina , Proteínas de Ligação a Telômeros/genética , Fator de Necrose Tumoral alfa/genética
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