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1.
PLoS Genet ; 7(7): e1002148, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21779174

RESUMO

RAD51 recombinase polymerizes at the site of double-strand breaks (DSBs) where it performs DSB repair. The loss of RAD51 causes extensive chromosomal breaks, leading to apoptosis. The polymerization of RAD51 is regulated by a number of RAD51 mediators, such as BRCA1, BRCA2, RAD52, SFR1, SWS1, and the five RAD51 paralogs, including XRCC3. We here show that brca2-null mutant cells were able to proliferate, indicating that RAD51 can perform DSB repair in the absence of BRCA2. We disrupted the BRCA1, RAD52, SFR1, SWS1, and XRCC3 genes in the brca2-null cells. All the resulting double-mutant cells displayed a phenotype that was very similar to that of the brca2-null cells. We suggest that BRCA2 might thus serve as a platform to recruit various RAD51 mediators at the appropriate position at the DNA-damage site.


Assuntos
Proteína BRCA2/genética , Epistasia Genética , Recombinação Homóloga , Rad51 Recombinase/genética , Animais , Camptotecina/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Galinhas , Aberrações Cromossômicas/efeitos dos fármacos , Aberrações Cromossômicas/efeitos da radiação , Cisplatino/farmacologia , Células Clonais , Dano ao DNA , Epistasia Genética/efeitos dos fármacos , Epistasia Genética/efeitos da radiação , Raios gama , Conversão Gênica/efeitos dos fármacos , Conversão Gênica/efeitos da radiação , Deleção de Genes , Loci Gênicos/genética , Genoma/genética , Recombinação Homóloga/efeitos dos fármacos , Recombinação Homóloga/efeitos da radiação , Modelos Biológicos , Fenótipo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/deficiência
2.
Proc Natl Acad Sci U S A ; 108(16): 6492-6, 2011 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-21464321

RESUMO

Interstrand cross-links (ICLs) block replication and transcription and thus are highly cytotoxic. In higher eukaryotes, ICLs processing involves the Fanconi anemia (FA) pathway and homologous recombination. Stalled replication forks activate the eight-subunit FA core complex, which ubiquitylates FANCD2-FANCI. Once it is posttranslationally modified, this heterodimer recruits downstream members of the ICL repairosome, including the FAN1 nuclease. However, ICL processing has been shown to also involve MUS81-EME1 and XPF-ERCC1, nucleases known to interact with SLX4, a docking protein that also can bind another nuclease, SLX1. To investigate the role of SLX4 more closely, we disrupted the SLX4 gene in avian DT40 cells. SLX4 deficiency caused cell death associated with extensive chromosomal aberrations, including a significant fraction of isochromatid-type breaks, with sister chromatids broken at the same site. SLX4 thus appears to play an essential role in cell proliferation, probably by promoting the resolution of interchromatid homologous recombination intermediates. Because ubiquitylation plays a key role in the FA pathway, and because the N-terminal region of SLX4 contains a ubiquitin-binding zinc finger (UBZ) domain, we asked whether this domain is required for ICL processing. We found that SLX4(-/-) cells expressing UBZ-deficient SLX4 were selectively sensitive to ICL-inducing agents, and that the UBZ domain was required for interaction of SLX4 with ubiquitylated FANCD2 and for its recruitment to DNA-damage foci generated by ICL-inducing agents. Our findings thus suggest that ubiquitylated FANCD2 recruits SLX4 to DNA damage sites, where it mediates the resolution of recombination intermediates generated during the processing of ICLs.


Assuntos
Reparo do DNA/fisiologia , Endonucleases/metabolismo , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Recombinases/metabolismo , Recombinação Genética/fisiologia , Ubiquitinação/fisiologia , Animais , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Galinhas , Cromátides/genética , Cromátides/metabolismo , Aberrações Cromossômicas/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/fisiologia , Reparo do DNA/efeitos dos fármacos , Endonucleases/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Recombinases/genética , Recombinação Genética/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos , Dedos de Zinco
3.
Cancer Res ; 81(18): 4861-4873, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34333454

RESUMO

Isocitrate dehydrogenase-mutant low-grade gliomas (IDHmut-LGG) grow slowly but frequently undergo malignant transformation, which eventually leads to premature death. Chemotherapy and radiotherapy treatments prolong survival, but can also induce genetic (or epigenetic) alterations involved in transformation. Here, we developed a mathematical model of tumor progression based on serial tumor volume data and treatment history of 276 IDHmut-LGGs classified by chromosome 1p/19q codeletion (IDHmut/1p19qcodel and IDHmut/1p19qnoncodel) and performed genome-wide mutational analyses, including targeted sequencing and longitudinal whole-exome sequencing data. These analyses showed that tumor mutational burden correlated positively with malignant transformation rate, and chemotherapy and radiotherapy significantly suppressed tumor growth but increased malignant transformation rate per cell by 1.8 to 2.8 times compared with before treatment. This model revealed that prompt adjuvant chemoradiotherapy prolonged malignant transformation-free survival in small IDHmut-LGGs (≤ 50 cm3). Furthermore, optimal treatment differed according to genetic alterations for large IDHmut-LGGs (> 50 cm3); adjuvant therapies delayed malignant transformation in IDHmut/1p19qnoncodel but often accelerated it in IDHmut/1p19qcodel. Notably, PI3K mutation was not associated with malignant transformation but increased net postoperative proliferation rate and decreased malignant transformation-free survival, prompting the need for adjuvant therapy in IDHmut/1p19qcodel. Overall, this model uncovered therapeutic strategies that could prevent malignant transformation and, consequently, improve overall survival in patients with IDHmut-LGGs. SIGNIFICANCE: A mathematical model successfully estimates malignant transformation-free survival and reveals a link between genetic alterations and progression, identifying precision medicine approaches for optimal treatment of IDH-mutant low-grade gliomas.


Assuntos
Transformação Celular Neoplásica/genética , Análise Mutacional de DNA/métodos , Glioma/genética , Glioma/patologia , Isocitrato Desidrogenase/genética , Modelos Teóricos , Mutação , Adulto , Biomarcadores Tumorais , Variações do Número de Cópias de DNA , Gerenciamento Clínico , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Glioma/mortalidade , Glioma/terapia , Humanos , Masculino , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Prognóstico , Resultado do Tratamento , Carga Tumoral
4.
JCO Clin Cancer Inform ; 3: 1-11, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30901235

RESUMO

Despite recent progress in diagnostic and multimodal treatment approaches, most cancer deaths are still caused by metastatic spread and the subsequent growth of tumor cells in sites distant from the primary organ. So far, few quantitative studies are available that allow for the estimation of metastatic parameters and the evaluation of alternative treatment strategies. Most computational studies have focused on situations in which the tumor cell population expands exponentially over time; however, tumors may eventually be subject to resource and space limitations so that their growth patterns deviate from exponential growth to adhere to density-dependent growth models. In this study, we developed a stochastic evolutionary model of cancer progression that considers alterations in metastasis-related genes and intercellular growth competition leading to density effects described by logistic growth. Using this stochastic model, we derived analytical approximations for the time between the initiation of tumorigenesis and diagnosis, the expected number of metastatic sites, the total number of metastatic cells, the size of the primary tumor, and survival. Furthermore, we investigated the effects of drug administration and surgical resection on these quantities and predicted outcomes for different treatment regimens. Parameter values used in the analysis were estimated from data obtained from a pancreatic cancer rapid autopsy program. Our theoretical approach allows for flexible modeling of metastatic progression dynamics.


Assuntos
Evolução Clonal , Modelos Biológicos , Neoplasias Pancreáticas/patologia , Algoritmos , Evolução Clonal/genética , Progressão da Doença , Humanos , Modelos Logísticos , Metástase Neoplásica , Neoplasias Pancreáticas/etiologia , Neoplasias Pancreáticas/mortalidade , Prognóstico , Processos Estocásticos
5.
PLoS One ; 14(4): e0215409, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31026288

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) exhibits a variety of phenotypes with regard to disease progression and treatment response. This variability complicates clinical decision-making despite the improvement of survival due to the recent introduction of FOLFIRINOX (FFX) and nab-paclitaxel. Questions remain as to the timing and sequence of therapies and the role of radiotherapy for unresectable PDAC. Here we developed a computational analysis platform to investigate the dynamics of growth, metastasis and treatment response to FFX, gemcitabine (GEM), and GEM+nab-paclitaxel. Our approach was informed using data of 1,089 patients treated at the Massachusetts General Hospital and validated using an independent cohort from Osaka Medical College. Our framework establishes a logistic growth pattern of PDAC and defines the Local Advancement Index (LAI), which determines the eventual primary tumor size and predicts the number of metastases. We found that a smaller LAI leads to a larger metastatic burden. Furthermore, our analyses ascertain that i) radiotherapy after induction chemotherapy improves survival in cases receiving induction FFX or with larger LAI, ii) neoadjuvant chemotherapy improves survival in cases with resectable PDAC, and iii) temporary cessations of chemotherapies do not impact overall survival, which supports the feasibility of treatment holidays for patients with FFX-associated adverse effects. Our findings inform clinical decision-making for PDAC patients and allow for the rational design of clinical strategies using FFX, GEM, GEM+nab-paclitaxel, neoadjuvant chemotherapy, and radiation.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/terapia , Desoxicitidina/análogos & derivados , Modelos Biológicos , Neoplasias Pancreáticas/terapia , Idoso , Albuminas/uso terapêutico , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Quimiorradioterapia/métodos , Tomada de Decisão Clínica , Simulação por Computador , Desoxicitidina/uso terapêutico , Progressão da Doença , Intervalo Livre de Doença , Esquema de Medicação , Estudos de Viabilidade , Feminino , Fluoruracila/uso terapêutico , Humanos , Irinotecano/uso terapêutico , Estimativa de Kaplan-Meier , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante/métodos , Oxaliplatina/uso terapêutico , Paclitaxel/uso terapêutico , Pâncreas/patologia , Pâncreas/cirurgia , Pancreatectomia , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Indução de Remissão/métodos , Carga Tumoral , Gencitabina
6.
Nat Cell Biol ; 21(7): 879-888, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31263265

RESUMO

Most human tumours are heterogeneous, composed of cellular clones with different properties present at variable frequencies. Highly heterogeneous tumours have poor clinical outcomes, yet the underlying mechanism remains poorly understood. Here, we show that minor subclones of breast cancer cells expressing IL11 and FIGF (VEGFD) cooperate to promote metastatic progression and generate polyclonal metastases composed of driver and neutral subclones. Expression profiling of the epithelial and stromal compartments of monoclonal and polyclonal primary and metastatic lesions revealed that this cooperation is indirect, mediated through the local and systemic microenvironments. We identified neutrophils as a leukocyte population stimulated by the IL11-expressing minor subclone and showed that the depletion of neutrophils prevents metastatic outgrowth. Single-cell RNA-seq of CD45+ cell populations from primary tumours, blood and lungs demonstrated that IL11 acts on bone-marrow-derived mesenchymal stromal cells, which induce pro-tumorigenic and pro-metastatic neutrophils. Our results indicate key roles for non-cell-autonomous drivers and minor subclones in metastasis.


Assuntos
Neoplasias da Mama/patologia , Neoplasias Pulmonares/patologia , Metástase Neoplásica/patologia , Neutrófilos/metabolismo , Microambiente Tumoral , Animais , Carcinogênese/metabolismo , Progressão da Doença , Humanos , Pulmão/patologia , Neoplasias Pulmonares/secundário , Células-Tronco Mesenquimais/citologia
8.
Cancer Cell ; 34(6): 939-953.e9, 2018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30472020

RESUMO

Members of the KDM5 histone H3 lysine 4 demethylase family are associated with therapeutic resistance, including endocrine resistance in breast cancer, but the underlying mechanism is poorly defined. Here we show that genetic deletion of KDM5A/B or inhibition of KDM5 activity increases sensitivity to anti-estrogens by modulating estrogen receptor (ER) signaling and by decreasing cellular transcriptomic heterogeneity. Higher KDM5B expression levels are associated with higher transcriptomic heterogeneity and poor prognosis in ER+ breast tumors. Single-cell RNA sequencing, cellular barcoding, and mathematical modeling demonstrate that endocrine resistance is due to selection for pre-existing genetically distinct cells, while KDM5 inhibitor resistance is acquired. Our findings highlight the importance of cellular phenotypic heterogeneity in therapeutic resistance and identify KDM5A/B as key regulators of this process.


Assuntos
Neoplasias da Mama/genética , Resistencia a Medicamentos Antineoplásicos/genética , Histona Desmetilases com o Domínio Jumonji/genética , Proteínas Nucleares/genética , Proteínas Repressoras/genética , Proteína 2 de Ligação ao Retinoblastoma/genética , Transcriptoma/genética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Feminino , Fulvestranto/farmacologia , Heterogeneidade Genética , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células MCF-7 , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Proteína 2 de Ligação ao Retinoblastoma/metabolismo , Transcriptoma/efeitos dos fármacos , Sequenciamento do Exoma/métodos
9.
Cancer Res ; 77(12): 3325-3335, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28381541

RESUMO

Phenotypic diversity in pancreatic ductal adenocarcinoma (PDAC) results in a variety of treatment responses. Rapid autopsy studies have revealed a subgroup of PDAC patients with a lower propensity to develop metastatic disease, challenging the common perception that all patients die of widely metastatic disease, but questions remain about root causes of this difference and the potential impact on treatment strategies. In this study, we addressed these questions through the development of a mathematical model of PDAC progression that incorporates the major alteration status of specific genes with predictive utility. The model successfully reproduced clinical outcomes regarding metastatic patterns and the genetic alteration status of patients from two independent cohorts from the United States and Japan. Using this model, we defined a candidate predictive signature in patients with low metastatic propensity. If a primary tumor contained a small fraction of cells with KRAS and additional alterations to CDKN2A, TP53, or SMAD4 genes, the patient was likely to exhibit low metastatic propensity. By using this predictive signature, we computationally simulated a set of clinical trials to model whether this subgroup would benefit from locally intensive therapies such as surgery or radiation therapy. The largest overall survival benefit resulted from complete resection, followed by adjuvant chemoradiation therapy and salvage therapies for isolated recurrence. While requiring prospective validation in a clinical trial, our results suggest a new tool to help personalize care in PDAC patients in seeking the most effective therapeutic modality for each individual. Cancer Res; 77(12); 3325-35. ©2017 AACR.


Assuntos
Carcinoma Ductal Pancreático/patologia , Modelos Teóricos , Neoplasias Pancreáticas/patologia , Medicina de Precisão/métodos , Transcriptoma , Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/terapia , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas
10.
Sci Rep ; 6: 34214, 2016 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-27694914

RESUMO

Although the capacity of the liver to recover its size after resection has enabled extensive liver resection, post-hepatectomy liver failure remains one of the most lethal complications of liver resection. Therefore, it is clinically important to discover reliable predictive factors after resection. In this study, we established a novel mathematical framework which described post-hepatectomy liver regeneration in each patient by incorporating quantitative clinical data. Using the model fitting to the liver volumes in series of computed tomography of 123 patients, we estimated liver regeneration rates. From the estimation, we found patients were divided into two groups: i) patients restored the liver to its original size (Group 1, n = 99); and ii) patients experienced a significant reduction in size (Group 2, n = 24). From discriminant analysis in 103 patients with full clinical variables, the prognosis of patients in terms of liver recovery was successfully predicted in 85-90% of patients. We further validated the accuracy of our model prediction using a validation cohort (prediction = 84-87%, n = 39). Our interdisciplinary approach provides qualitative and quantitative insights into the dynamics of liver regeneration. A key strength is to provide better prediction in patients who had been judged as acceptable for resection by current pragmatic criteria.


Assuntos
Regeneração Hepática , Modelos Estatísticos , Período Pós-Operatório , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade
11.
Sci Rep ; 5: 15886, 2015 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-26515895

RESUMO

Metastasis is a leading cause of cancer-related deaths. Carcinoma generally initiates at a specific organ as a primary tumor, but eventually metastasizes and forms tumor sites in other organs. In this report, we developed a mathematical model of cancer progression with alterations in metastasis-related genes. In cases in which tumor cells acquire metastatic ability through two steps of genetic alterations, we derive formulas for the probability, the expected number, and the distribution of the number of metastases. Moreover, we investigate practical pancreatic cancer disease progression in cases in which both one and two steps of genetic alterations are responsible for metastatic formation. Importantly, we derive a mathematical formula for the survival outcome validated using clinical data as well as direct simulations. Our model provides theoretical insights into how invisible metastases distribute upon diagnosis with respect to growth rates, (epi)genetic alteration rates, metastatic rate, and detection size. Prediction of survival outcome using the formula is of clinical importance in terms of determining therapeutic strategies.


Assuntos
Modelos Teóricos , Metástase Neoplásica , Neoplasias Pancreáticas/patologia , Progressão da Doença , Epigênese Genética , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Análise de Sobrevida
12.
Cell Rep ; 11(1): 71-84, 2015 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-25843711

RESUMO

Upon systemic bacterial infection, hematopoietic stem and progenitor cells (HSPCs) migrate to the periphery in order to supply a sufficient number of immune cells. Although pathogen-associated molecular patterns reportedly mediate HSPC activation, how HSPCs detect pathogen invasion in vivo remains elusive. Bacteria use the second messenger bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) for a variety of activities. Here, we report that c-di-GMP comprehensively regulated both HSPCs and their niche cells through an innate immune sensor, STING, thereby inducing entry into the cell cycle and mobilization of HSPCs while decreasing the number and repopulation capacity of long-term hematopoietic stem cells. Furthermore, we show that type I interferon acted as a downstream target of c-di-GMP to inhibit HSPC expansion in the spleen, while transforming growth factor-ß was required for c-di-GMP-dependent splenic HSPC expansion. Our results define machinery underlying the dynamic regulation of HSPCs and their niches during bacterial infection through c-di-GMP/STING signaling.


Assuntos
GMP Cíclico/análogos & derivados , Células-Tronco Hematopoéticas/metabolismo , Imunidade Inata , Fator Regulador 3 de Interferon/genética , Proteínas de Membrana/genética , Animais , Bactérias/metabolismo , Bactérias/patogenicidade , GMP Cíclico/administração & dosagem , GMP Cíclico/imunologia , GMP Cíclico/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/microbiologia , Fator Regulador 3 de Interferon/imunologia , Proteínas de Membrana/imunologia , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Nicho de Células-Tronco/imunologia , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/imunologia
13.
PLoS One ; 9(8): e105724, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25158060

RESUMO

Platinum drugs and PARP inhibitors ("PARPis") are considered to be effective in BRCA-associated cancers with impaired DNA repair. These agents cause stalled and collapsed replication forks and create double-strand breaks effectively in the absence of repair mechanisms, resulting in arrest of the cell cycle and induction of cell death. However, recent studies have shown failure of these chemotherapeutic agents due to emerging drug resistance. In this study, we developed a stochastic model of BRCA-associated cancer progression in which there are four cancer populations: those with (i) functional BRCA, (ii) dysfunctional BRCA, (iii) functional BRCA and a growth advantage, and (iv) dysfunctional BRCA and a growth advantage. These four cancer populations expand from one cancer cell with normal repair function until the total cell number reaches a detectable amount. We derived formulas for the probability and expected numbers of each population at the time of detection. Furthermore, we extended the model to consider the tumor dynamics during treatment. Results from the model were validated and showed good agreement with clinical and experimental evidence in BRCA-associated cancers. Based on the model, we investigated conditions in which drug resistance during the treatment course originated from either a pre-existing drug-resistant population or a de novo population, due to secondary mutations. Finally, we found that platinum drugs and PARPis were effective if (i) BRCA inactivation is present, (ii) the cancer was diagnosed early, and (iii) tumor growth is rapid. Our results indicate that different types of cancers have a preferential way of acquiring resistance to platinum drugs and PARPis according to their growth and mutational characteristics.


Assuntos
Antineoplásicos/farmacologia , Carboplatina/farmacologia , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Progressão da Doença , Evolução Molecular , Genes BRCA1 , Genes BRCA2 , Humanos , Modelos Biológicos , Neoplasias/genética , Neoplasias/patologia
15.
PLoS One ; 8(4): e60043, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23573231

RESUMO

Morphological analysis of mitotic chromosomes is used to detect mutagenic chemical compounds and to estimate the dose of ionizing radiation to be administered. It has long been believed that chromosomal breaks are always associated with double-strand breaks (DSBs). We here provide compelling evidence against this canonical theory. We employed a genetic approach using two cell lines, chicken DT40 and human Nalm-6. We measured the number of chromosomal breaks induced by three replication-blocking agents (aphidicolin, 5-fluorouracil, and hydroxyurea) in DSB-repair-proficient wild-type cells and cells deficient in both homologous recombination and nonhomologous end-joining (the two major DSB-repair pathways). Exposure of cells to the three replication-blocking agents for at least two cell cycles resulted in comparable numbers of chromosomal breaks for RAD54(-/-/)KU70(-/-) DT40 clones and wild-type cells. Likewise, the numbers of chromosomal breaks induced in RAD54(-/-/)LIG4(-/-) Nalm-6 clones and wild-type cells were also comparable. These data indicate that the replication-blocking agents can cause chromosomal breaks unassociated with DSBs. In contrast with DSB-repair-deficient cells, chicken DT40 cells deficient in PIF1 or ATRIP, which molecules contribute to the completion of DNA replication, displayed higher numbers of mitotic chromosomal breaks induced by aphidicolin than did wild-type cells, suggesting that single-strand gaps left unreplicated may result in mitotic chromosomal breaks.


Assuntos
Quebra Cromossômica , Replicação do DNA/efeitos dos fármacos , Mitose , Animais , Antígenos Nucleares/genética , Afidicolina/farmacologia , Apoptose , Linhagem Celular Tumoral , Galinhas , Quebras de DNA de Cadeia Dupla , DNA Helicases/fisiologia , Reparo do DNA , Enzimas Reparadoras do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Fluoruracila/farmacologia , Técnicas de Inativação de Genes , Humanos , Hidroxiureia/farmacologia , Autoantígeno Ku
16.
Environ Mol Mutagen ; 52(7): 547-61, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21538559

RESUMO

Included among the quantitative high throughput screens (qHTS) conducted in support of the US Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in seven isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis.


Assuntos
Dano ao DNA/efeitos dos fármacos , Distúrbios no Reparo do DNA/genética , Mutagênicos/toxicidade , Acetamidas/toxicidade , Animais , Linhagem Celular , Galinhas , Aberrações Cromossômicas , Dano ao DNA/genética , Dactinomicina/toxicidade , Doxorrubicina/toxicidade , Lovastatina/toxicidade , Melfalan/toxicidade , Testes de Mutagenicidade , Resorcinóis/toxicidade , Tolueno/análogos & derivados , Tolueno/toxicidade
17.
DNA Repair (Amst) ; 9(12): 1292-8, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21030320

RESUMO

Chemicals used industrially and commercially are required by law to be assessed for their genotoxic potential. However, all currently used assays have major limitations and despite intense effort, there is no universal agreement on which tests should be employed, or how to interpret results. We have developed a new assay system using the chicken DT40 B cell line that offers a number of significant advantages over current methodologies. Our assay could provide enhanced sensitivity using genetically defined and phenotypically characterized mutants defective in DNA repair pathways. Furthermore, analysis of the mutants, using DNA repair proficient wild-type cells as a negative control, minimizes false negative outcomes. Assessing the different responses of a panel of mutants representative of all repair pathways, mechanistic detail of genotoxicity can be determined. This unique feature, as well as reducing the false positive rate, strengthens positive identifications and is useful when extrapolating results to the human context. Our panel of mutants is likely to be useful in screening large compound libraries for an emerging class of chemotherapeutic drugs, which includes inhibitors of DNA repair enzymes such as PARP and DNA polymerases.


Assuntos
Dano ao DNA , Reparo do DNA/genética , Replicação do DNA/fisiologia , Testes de Mutagenicidade/métodos , Mutagênicos/metabolismo , Mutação/genética , Animais , Linhagem Celular , Galinhas
18.
Biol Trace Elem Res ; 130(3): 273-82, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19221697

RESUMO

Some of the rare earth elements such as Sc are believed to be non-toxic and, at present, are widely utilized for the replacement of toxic heavy metals in technological applications, but they are not entirely free of toxicity, with hidden potential health risks. In this animal experiment, we report the urinary scandium (Sc) excretion rate and nephrotoxiciy in male Wistar rats. For this purpose, the rats were given a single dose of a solution of scandium chloride by intraperitoneal injection. The Sc excretion (U-Sc) was determined in 24-h urine samples by inductively coupled plasma-argon emission spectrometry along with the Sc nephrotoxicity, urine volume (UV), creatinine (Crt), beta-2-microglobulin (beta2-MG) and N-acetyl-beta-D-glucosaminidase (NAG). A dose-dependent Sc excretion of 0.0063% (r = 0.97) via 24-h urine was confirmed. The administration of Sc induced a significant decrease of UV and Crt and a significant increase of NAG and beta2-MG. These results suggest that U-Sc can be a useful tool for monitoring Sc exposure. The formation of Sc colloidal conjugates that deposit in glomeruli may be the cause of a reduction of the glomerular filtration rate. We propose that the analytical method and results described in this study will be of great importance for future toxicological studies on Sc exposure.


Assuntos
Cloretos/toxicidade , Rim/efeitos dos fármacos , Escândio/urina , Acetilglucosaminidase/urina , alfa-Globulinas/urina , Animais , Cloretos/administração & dosagem , Creatinina/urina , Exposição Ambiental/análise , Injeções Intraperitoneais , Rim/patologia , Masculino , Ratos , Ratos Wistar , Escândio/administração & dosagem , Escândio/normas , Escândio/toxicidade , Espectrofotometria Atômica/métodos , Microglobulina beta-2/urina
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