RESUMO
H2A.J is a poorly studied mammalian-specific variant of histone H2A. We used immunohistochemistry to study its localization in various human and mouse tissues. H2A.J showed cell-type specific expression with a striking enrichment in luminal epithelial cells of multiple glands including those of breast, prostate, pancreas, thyroid, stomach, and salivary glands. H2A.J was also highly expressed in many carcinoma cell lines and in particular, those derived from luminal breast and prostate cancer. H2A.J thus appears to be a novel marker for luminal epithelial cancers. Knocking-out the H2AFJ gene in T47D luminal breast cancer cells reduced the expression of several estrogen-responsive genes which may explain its putative tumorigenic role in luminal-B breast cancer.
Assuntos
Glândulas Endócrinas/metabolismo , Células Epiteliais/metabolismo , Histonas/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Glândulas Endócrinas/patologia , Células Epiteliais/patologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Variação Genética , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos/genética , Gravidez , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologiaRESUMO
Phosphorylation of histone H2AX is a major contributor to efficient DNA repair. We recently reported neurobehavioral deficits in mice lacking H2AX. Here we establish that this neural failure stems from impairment of mitochondrial function and repression of the mitochondrial biogenesis gene PGC-1α. H2AX loss leads to reduced levels of the major subunits of the mitochondrial respiratory complexes in mouse embryonic fibroblasts and in the striatum, a brain region particularly vulnerable to mitochondrial damage. These defects are substantiated by disruption of the mitochondrial shape in H2AX mutant cells. Ectopic expression of PGC-1α restores mitochondrial oxidative phosphorylation complexes and mitigates cell death. H2AX knockout mice display increased neuronal death in the brain when challenged with 3-nitropronionic acid, which targets mitochondria. This study establishes a role for H2AX in mitochondrial homeostasis associated with neuroprotection.
Assuntos
Histonas/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/metabolismo , Fosforilação Oxidativa , Animais , Morte Celular , Transporte de Elétrons/fisiologia , Histonas/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Células-Tronco Neurais/citologia , Neurônios/citologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , FosforilaçãoRESUMO
ATM drives DNA repair by phosphorylating the histone variant H2AX. While ATM mutations elicit prominent neurobehavioral phenotypes, neural roles for H2AX have been elusive. We report impaired motor learning and balance in H2AX-deficient mice. Mitigation of reactive oxygen species (ROS) with N-acetylcysteine (NAC) reverses the behavioral deficits. Mouse embryonic fibroblasts deficient for H2AX exhibit increased ROS production and failure to activate the antioxidant response pathway controlled by the transcription factor NRF2. The NRF2 targets GCLC and NQO1 are depleted in the striatum of H2AX knockouts, one of the regions most vulnerable to ROS-mediated damage. These findings establish a role for ROS in the behavioral deficits of H2AX knockout mice and reveal a physiologic function of H2AX in mediating influences of oxidative stress on NRF2-transcriptional targets and behavior.
Assuntos
Comportamento Animal , Histonas/deficiência , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo , Acetilcisteína/química , Animais , Antioxidantes/química , Corpo Estriado/metabolismo , Dano ao DNA , Fibroblastos/metabolismo , Células HEK293 , Heterozigoto , Histonas/fisiologia , Humanos , Camundongos , Camundongos Knockout , Microscopia Confocal , Modelos Neurológicos , Destreza Motora , Oxirredução , Fenótipo , Fosforilação , Espécies Reativas de Oxigênio/metabolismoRESUMO
The standard-of-care for advanced small cell lung cancer (SCLC) is chemotherapy with cisplatin+etoposide (C+E). Most patients have chemosensitive disease at the outset, but disease frequently relapses and limits survival. Efforts to improve therapeutic outcomes in SCLC and other neuroendocrine cancers have focused on epigenetic agents, including the histone deacetylase inhibitor belinostat. The primary objective was to determine the maximum tolerated dose of the combination of belinostat (B) with C+E. Belinostat was administered as a 48-h continuous intravenous infusion on days 1-2; cisplatin was administered as a 1-h intravenous infusion on day 2; and etoposide was administered as a 1-h intravenous infusion on days 2, 3, and 4. Twenty-eight patients were recruited in this single-center study. The maximum tolerated dose was belinostat 500 mg/m/24 h, cisplatin 60 mg/m, and etoposide 80 mg/m. The combination was safe, although some patients were more susceptible to adverse events. Hematologic toxicities were most commonly observed. Objective responses were observed in 11 (39%) of 28 patients and seven (47%) of 15 patients with neuroendocrine tumors (including SCLC). Patients carrying more than three copies of variant UGT1A1 (*28 and *60) had higher serum levels of belinostat because of slower clearance. DNA damage peaked at 36 h after the initiation of belinostat, as did global lysine acetylation, but returned to baseline 12 h after the end of infusion. The combination of B+C+E is safe and active in SCLC and other neuroendocrine cancers. Future phase II studies should consider genotyping patients for UGT1A1*28 and UGT1A1*60 and to identify patients at an increased risk of adverse events.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Tumores Neuroendócrinos/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Cisplatino/administração & dosagem , Etoposídeo/administração & dosagem , Feminino , Glucuronosiltransferase/genética , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/uso terapêutico , Histonas/sangue , Humanos , Ácidos Hidroxâmicos/administração & dosagem , Infusões Intravenosas , Neoplasias Pulmonares/patologia , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Tumores Neuroendócrinos/patologia , Carcinoma de Pequenas Células do Pulmão/patologia , Sulfonamidas/administração & dosagem , Resultado do TratamentoRESUMO
Recent integrative epigenome analyses highlight the importance of functionally distinct chromatin states for accurate cell function. How these states are established and maintained is a matter of intense investigation. Here, we present evidence for DNA damage as an unexpected means to shape a protective chromatin environment at regions of recurrent replication stress (RS). Upon aberrant fork stalling, DNA damage signaling and concomitant H2AX phosphorylation coordinate the FACT-dependent deposition of macroH2A1.2, a histone variant that promotes DNA repair by homologous recombination (HR). MacroH2A1.2, in turn, facilitates the accumulation of the tumor suppressor and HR effector BRCA1 at replication forks to protect from RS-induced DNA damage. Consequently, replicating primary cells steadily accrue macroH2A1.2 at fragile regions, whereas macroH2A1.2 loss in these cells triggers DNA damage signaling-dependent senescence, a hallmark of RS. Altogether, our findings demonstrate that recurrent DNA damage contributes to the chromatin landscape to ensure the epigenomic integrity of dividing cells.
Assuntos
Carcinogênese/genética , Cromatina/genética , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Histonas/genética , Recombinação Homóloga/genética , Proteína BRCA1/metabolismo , Divisão Celular/genética , Células Cultivadas , Senescência Celular/genética , Instabilidade Genômica/fisiologia , Humanos , Transdução de Sinais/genéticaRESUMO
The senescence of mammalian cells is characterized by a proliferative arrest in response to stress and the expression of an inflammatory phenotype. Here we show that histone H2A.J, a poorly studied H2A variant found only in mammals, accumulates in human fibroblasts in senescence with persistent DNA damage. H2A.J also accumulates in mice with aging in a tissue-specific manner and in human skin. Knock-down of H2A.J inhibits the expression of inflammatory genes that contribute to the senescent-associated secretory phenotype (SASP), and over expression of H2A.J increases the expression of some of these genes in proliferating cells. H2A.J accumulation may thus promote the signalling of senescent cells to the immune system, and it may contribute to chronic inflammation and the development of aging-associated diseases.
Assuntos
Senescência Celular/genética , Citocinas/genética , Histonas/genética , Fatores Etários , Animais , Linhagem Celular , Proliferação de Células/genética , Citocinas/metabolismo , Dano ao DNA , Fibroblastos/citologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Variação Genética , Histonas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Pele/metabolismoRESUMO
Upon DNA damage or other stressors, the tumor suppressor p53 is activated, leading to transient expression of the cyclin-dependent kinase inhibitor (CKI) p21. This either triggers momentary G1 cell cycle arrest or leads to a chronic state of senescence or apoptosis, a form of genome guardianship. In the clinic, the presence of p21 has been considered an indicator of wildtype p53 activity. However, recent evidence suggests that p21 also acts as an oncogenic factor in a p53-deficient environment. Here, we discuss the controversial aspects of the two-faced involvement of p21 in cancer and speculate on how this new information may increase our understanding of its role in cancer pathogenesis. Prevailing notions indicate that p21 might also act as antiapoptotic agent, which may have relevant implications for future therapeutic strategies.
Assuntos
Apoptose , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Inibidor de Quinase Dependente de Ciclina p21/genética , Dano ao DNA , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genéticaRESUMO
The Fukushima Daiichi Nuclear Power Plant (FNPP) accident, the largest nuclear incident since the 1986 Chernobyl disaster, occurred when the plant was hit by a tsunami triggered by the Great East Japan Earthquake on March 11, 2011. The subsequent uncontrolled release of radioactive substances resulted in massive evacuations in a 20-km zone. To better understand the biological consequences of the FNPP accident, we have been measuring DNA damage levels in cattle in the evacuation zone. DNA damage was evaluated by assessing the levels of DNA double-strand breaks in peripheral blood lymphocytes by immunocytofluorescence-based quantification of γ-H2AX foci. A greater than two-fold increase in the fraction of damaged lymphocytes was observed in all animal cohorts within the evacuation zone, and the levels of DNA damage decreased slightly over the 700-day sample collection period. While the extent of damage appeared to be independent of the distance from the accident site and the estimated radiation dose from radiocesium, we observed age-dependent accumulation of DNA damage. Thus, this study, which was the first to evaluate the biological impact of the FNPP accident utilizing the γ-H2AX assays, indicated the causal relation between high levels of DNA damage in animals living in the evacuation zone and the FNPP accident.
Assuntos
Dano ao DNA/genética , Acidente Nuclear de Fukushima , Linfócitos/fisiologia , Linfócitos/efeitos da radiação , Exposição à Radiação/análise , Monitoramento de Radiação/métodos , Animais , Bioensaio/métodos , Bovinos , Causalidade , Células Cultivadas , Relação Dose-Resposta à Radiação , Feminino , Doses de Radiação , Sensibilidade e EspecificidadeRESUMO
Phosphorylated H2AX (γ-H2AX) is a sensitive marker for DNA double-strand breaks (DSBs), but the variability of H2AX expression in different cell and tissue types makes it difficult to interpret the meaning of the γ-H2AX level. Furthermore, the assays commonly used for γ-H2AX detection utilize laborious and low-throughput microscopy-based methods. We describe here an ELISA assay that measures both phosphorylated H2AX and total H2AX absolute amounts to determine the percentage of γ-H2AX, providing a normalized value representative of the amount of DNA damage. We demonstrate the utility of the assay to measure DSBs introduced by either ionizing radiation or DNA-damaging agents in cultured cells and in xenograft models. Furthermore, utilizing the NCI-60 cancer cell line panel, we show a correlation between the basal fraction of γ-H2AX and cellular mutation levels. This additional application highlights the ability of the assay to measure γ-H2AX levels in many extracts at once, making it possible to correlate findings with other cellular characteristics. Overall, the γ-H2AX ELISA represents a novel approach to quantifying DNA damage, which may lead to a better understanding of mutagenic pathways in cancer and provide a useful biomarker for monitoring the effectiveness of DNA-damaging anticancer agents.
Assuntos
Bioensaio/métodos , Dano ao DNA/genética , Histonas/metabolismo , Fosforilação/fisiologia , Animais , Linhagem Celular Tumoral , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Feminino , Histonas/genética , Humanos , Camundongos , Camundongos Nus , MutaçãoRESUMO
The development of novel cancer treatments is a challenging task, partly because results from model systems often fail to predict drug efficacy in humans, and also tumors are often inaccessible for biochemical analysis, preventing effective monitoring of drug activity in vivo. Utilizing a model system, we evaluated the use of drug-induced DNA damage in surrogate tissues as indicators of drug efficacy. Samples of a commercially available melanoma skin model (Mattek MLNM-FT-A375) containing keratinocyte and fibroblast layers with melanoma nodules were subjected to various chemotherapeutic regimens for one, four, or eight days. At these times they were analyzed for DNA double-stranded breaks (γH2AX foci) and apoptosis (TUNEL). A wide range of drug responses in both tumor and normal tissues were observed and cataloged. For the melanoma, the most common drug response was apoptosis. The basal keratinocyte layer, which was the most reliable indicator of drug response in the melanoma skin model, responded with γH2AX foci formation that was abrupt and transient. The relationships between tumor and surrogate tissue drug responses are complex, indicating that while surrogate tissue drug responses may be useful clinical tools, careful control of variables such as the timing of sampling may be important in interpreting the results.
Assuntos
Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Apoptose/efeitos dos fármacos , Dano ao DNA , DNA de Neoplasias/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Queratinócitos/efeitos dos fármacos , Melanoma/genética , Melanoma/patologia , Mutagênicos/farmacologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
The epithelial-mesenchymal transition (EMT) is thought to be essential for cancer metastasis. While chromatin remodeling is involved in EMT, which processes contribute to this remodeling remain poorly investigated. Recently, we showed that silencing or removal of the histone variant H2A.X induced mesenchymal-like characteristics, including activation of the EMT transcription factors, Slug and Zeb1 in human colon cancer cells. Here, we provide the evidence that H2A.X loss in human non-tumorigenic breast cell line MCF10A results in a robust EMT activation, as substantiated by a genome-wide expression analysis. Cells deficient for H2A.X exhibit enhanced migration and invasion, along with an activation of a set of mesenchymal genes and a concomitant repression of epithelial genes. In the breast model, the EMT-related transcription factor Twist1 cooperates with Slug to regulate EMT upon H2A.X Loss. Of interest, H2A.X expression level tightly correlates with Twist1, and to a lesser extent with Slug in the panel of human breast cancer cell lines of the NCI-60 datasets. These new findings indicate that H2A.X is involved in the EMT processes in cells of different origins but pairing with transcription factors for EMT may be tissue specific.
Assuntos
Mama/patologia , Transição Epitelial-Mesenquimal , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição da Família Snail/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Modelos BiológicosRESUMO
PURPOSE: Indenoisoquinolines are non-camptothecin topoisomerase I (TopI) inhibitors that overcome the limitations of camptothecins: chemical instability and camptothecin resistance. Two dosing schedules of the novel indenoisoquinoline, indotecan (LMP400), were evaluated in patients with advanced solid tumors. METHODS: The maximum tolerated dose (MTD), toxicities, and pharmacokinetics of two indotecan drug administration schedules (daily for 5 days or weekly) were investigated. Modulation of TopI and the phosphorylation of histone H2AX (γH2AX) were assayed in tumor biopsies; γH2AX levels were also evaluated in circulating tumor cells (CTCs) and hair follicles to assess DNA damage response. RESULTS: An MTD of 60 mg/m(2)/day was established for the daily regimen, compared to 90 mg/m(2) for the weekly regimen. The TopI response to drug showed target engagement in a subset of tumor biopsies. Pharmacokinetics profiles demonstrated a prolonged terminal half-life and tissue accumulation compared to topotecan. Dose-dependent decreases in total CTCs were measured in seven patients. Formation of γH2AX-positive foci in CTCs (day 3) and hair follicles (4-6 h) was observed following treatment. CONCLUSIONS: We established the MTD of two dosing schedules for a novel TopI inhibitor, indotecan. Target engagement was demonstrated as Top1 downregulation and γH2AX response. No objective responses were observed on either schedule in this small patient cohort. The principal toxicity of both schedules was myelosuppression; no significant gastrointestinal problems were observed. Increased DNA damage response was observed in CTCs, hair follicles, and a subset of tumor biopsies.
Assuntos
Benzodioxóis/administração & dosagem , DNA Topoisomerases Tipo I/metabolismo , Histonas/metabolismo , Isoquinolinas/administração & dosagem , Neoplasias/tratamento farmacológico , Inibidores da Topoisomerase I/administração & dosagem , Adulto , Idoso , Benzodioxóis/efeitos adversos , Benzodioxóis/farmacocinética , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Esquema de Medicação , Feminino , Folículo Piloso/metabolismo , Meia-Vida , Humanos , Isoquinolinas/efeitos adversos , Isoquinolinas/farmacocinética , Masculino , Dose Máxima Tolerável , Pessoa de Meia-Idade , Neoplasias/patologia , Fatores de Tempo , Distribuição Tecidual , Inibidores da Topoisomerase I/efeitos adversos , Inibidores da Topoisomerase I/farmacocinética , Topotecan/farmacocinética , Adulto JovemRESUMO
Purpose To examine the effect of breast shielding on blood lymphocyte deoxyribonucleic acid (DNA) double-strand-break levels resulting from in vivo radiation and ex vivo radiation at breast-tissue level, and the effect of breast shielding on image quality. Materials and Methods The study was approved by institutional review and commpliant with HIPAA guidelines. Adult women who underwent 64-section coronary computed tomographic (CT) angiography and who provided informed consent were prospectively randomized to the use (n = 50) or absence (n = 51) of bismuth breast shields. Peripheral blood samples were obtained before and 30 minutes after in vivo radiation during CT angiography to compare DNA double-strand-break levels by γ-H2AX immunofluorescence in blood lymphocytes. To estimate DNA double-strand-break induction at breast-tissue level, a blood sample was taped to the sternum for ex vivo radiation with or without shielding. Data were analyzed by linear regression and independent sample t tests. Results Breast shielding had no effect on DNA double-strand-break levels from ex vivo radiation of blood samples under shields at breast-tissue level (unadjusted regression: ß = .08; P = .43 versus no shielding), or in vivo radiation of circulating lymphocytes (ß = -.07; P = .50). Predictors of increased DNA double-strand-break levels included total radiation dose, increasing tube potential, and tube current (P < .05). With current radiation exposures (median, 3.4 mSv), breast shielding yielded a 33% increase in image noise and 19% decrease in the rate of excellent quality ratings. Conclusion Among women who underwent coronary CT angiography, breast shielding had no effect on DNA double-strand-break levels in blood lymphocytes exposed to in vivo radiation, or ex vivo radiation at breast-tissue level. At present relatively low radiation exposures, breast shielding contributed to an increase in image noise and a decline in image quality. The findings support efforts to minimize radiation by primarily optimizing CT settings. (©) RSNA, 2016 Clinical trial registration no. NCT02617888 Online supplemental material is available for this article.
Assuntos
Mama/efeitos da radiação , Angiografia por Tomografia Computadorizada , Angiografia Coronária , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Cardiopatias/diagnóstico por imagem , Feminino , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Doses de Radiação , Proteção Radiológica/métodos , Interpretação de Imagem Radiográfica Assistida por ComputadorRESUMO
The epithelial-mesenchymal transition (EMT), considered essential for metastatic cancer, has been a focus of much research, but important questions remain. Here, we show that silencing or removing H2A.X, a histone H2A variant involved in cellular DNA repair and robust growth, induces mesenchymal-like characteristics including activation of EMT transcription factors, Slug and ZEB1, in HCT116 human colon cancer cells. Ectopic H2A.X re-expression partially reverses these changes, as does silencing Slug and ZEB1. In an experimental metastasis model, the HCT116 parental and H2A.X-null cells exhibit a similar metastatic behaviour, but the cells with re-expressed H2A.X are substantially more metastatic. We surmise that H2A.X re-expression leads to partial EMT reversal and increases robustness in the HCT116 cells, permitting them to both form tumours and to metastasize. In a human adenocarcinoma panel, H2A.X levels correlate inversely with Slug and ZEB1 levels. Together, these results point to H2A.X as a regulator of EMT.
Assuntos
Adenocarcinoma/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Histonas/genética , Proteínas de Homeodomínio/genética , Metástase Neoplásica/genética , Fatores de Transcrição/genética , Animais , Western Blotting , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Imunofluorescência , Técnicas de Silenciamento de Genes , Variação Genética , Células HCT116 , Células HEK293 , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Transcrição da Família Snail , Homeobox 1 de Ligação a E-box em Dedo de ZincoRESUMO
Despite significant advances in the treatment of Hodgkin's lymphoma (HL), a significant proportion of patients will not respond or will subsequently relapse. We identified CD25, the IL-2 receptor alpha subunit, as a favorable target for systemic radioimmunotherapy of HL. The scientific basis for the clinical trial was that, although most normal cells with exception of Treg cells do not express CD25, it is expressed by a minority of Reed-Sternberg cells and by most polyclonal T cells rosetting around Reed-Sternberg cells. Forty-six patients with refractory and relapsed HL were evaluated with up to seven i.v. infusions of the radiolabeled anti-CD25 antibody (90)Y-daclizumab. (90)Y provides strong ß emissions that kill tumor cells at a distance by a crossfire effect. In 46 evaluable HL patients treated with (90)Y-daclizumab there were 14 complete responses and nine partial responses; 14 patients had stable disease, and nine progressed. Responses were observed both in patients whose Reed-Sternberg cells expressed CD25 and in those whose neoplastic cells were CD25(-) provided that associated rosetting T cells expressed CD25. As assessed using phosphorylated H2AX (γ-H2AX) as a bioindicator of the effects of radiation exposure, predominantly nonmalignant cells in the tumor microenvironment manifested DNA damage, as reflected by increased expression of γ-H2AX. Toxicities were transient bone-marrow suppression and myelodysplastic syndrome in six patients who had not been evaluated with bone-marrow karyotype analyses before therapy. In conclusion, repeated (90)Y-daclizumab infusions directed predominantly toward nonmalignant T cells rosetting around Reed-Sternberg cells provided meaningful therapy for select HL patients.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Doença de Hodgkin/tratamento farmacológico , Imunoglobulina G/uso terapêutico , Subunidade alfa de Receptor de Interleucina-2/imunologia , Radioisótopos de Ítrio/química , Adulto , Idoso , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/imunologia , Daclizumabe , Feminino , Doença de Hodgkin/imunologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Fosforilação , Recidiva , Adulto JovemRESUMO
Friedreich ataxia (FRDA) is a member of the Repeat Expansion Diseases, a group of genetic conditions resulting from an increase/expansion in the size of a specific tandem array. FRDA results from expansion of a GAA/TTC-tract in the first intron of the frataxin gene (FXN). The disease-associated tandem repeats all form secondary structures that are thought to contribute to the propensity of the repeat to expand. The subset of these diseases that result from a CGG/CCG-repeat expansion, such as Fragile X syndrome, also express a folate-sensitive fragile site coincident with the repeat on the affected chromosome. This chromosome fragility involves the generation of chromosome/chromatid gaps or breaks, or the high frequency loss of one or both copies of the affected gene when cells are grown under folate stress or as we showed previously, in the presence of an inhibitor of the ATM checkpoint kinase. Whether Repeat Expansion Disease loci containing different repeats form similar fragile sites was not known. We show here that the region of chromosome 9 that contains the FXN locus is intrinsically prone to breakage in vivo even in control cells. However, like FXS alleles, FRDA alleles show significantly elevated levels of chromosome abnormalities in the presence of an ATM inhibitor, consistent with the formation of a fragile site.
Assuntos
Fragilidade Cromossômica/genética , Cromossomos Humanos Par 9/genética , Ataxia de Friedreich/genética , Proteínas de Ligação ao Ferro/genética , Sequência de Bases , Linhagem Celular , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Análise de Sequência de DNA , Expansão das Repetições de Trinucleotídeos , FrataxinaRESUMO
Ataxia telangiectasia (A-T), a rare autosomal recessive disorder characterized by progressive cerebellar degeneration and a greatly increased incidence of cancer among other symptoms, is caused by a defective or missing ataxia telangiectasia mutated (ATM) gene. The ATM protein has roles in DNA repair and in the regulation of reactive oxygen species (ROS). Here, we provide, to our knowledge, the first evidence that NADPH oxidase 4 (NOX4) is involved in manifesting A-T disease. We showed that NOX4 expression levels are higher in A-T cells, and that ATM inhibition leads to increased NOX4 expression in normal cells. A-T cells exhibit elevated levels of oxidative DNA damage, DNA double-strand breaks and replicative senescence, all of which are partially abrogated by down-regulation of NOX4 with siRNA. Sections of degenerating cerebelli from A-T patients revealed elevated NOX4 levels. ATM-null mice exhibit A-T disease but they die from cancer before the neurological symptoms are manifested. Injecting Atm-null mice with fulvene-5, a specific inhibitor of NOX4 and NADPH oxidase 2 (NOX2), decreased their elevated cancer incidence to that of the controls. We conclude that, in A-T disease in humans and mice, NOX4 may be critical mediator and targeting it will open up new avenues for therapeutic intervention in neurodegeneration.
Assuntos
Ataxia Telangiectasia/enzimologia , NADPH Oxidases/metabolismo , Adulto , Animais , Ataxia Telangiectasia/patologia , Dano ao DNA , Replicação do DNA , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , NADPH Oxidase 4 , Adulto JovemRESUMO
Human exposure to ionizing radiation from medical procedures has increased sharply in the last three decades. Recent epidemiological studies suggest a direct relationship between exposure to ionizing radiation and health problems, including cancer incidence. Therefore, minimizing the impact of radiation exposure in patients has become a priority in the development of future clinical practices. Crucial players in radiation-induced DNA damage include reactive oxygen species (ROS), but the sources of these have remained elusive. To the best of our knowledge, we show here for the first time that two members of the ROS-generating NADPH oxidase family (NOXs), NOX4 and NOX5, are involved in radiation-induced DNA damage. Depleting these two NOXs in human primary fibroblasts resulted in reduced levels of DNA damage as measured by levels of radiation-induced foci, a marker of DNA double-strand breaks (DSBs) and the comet assay coupled with increased cell survival. NOX involvement was substantiated with fulvene-5, a NOXs-specific inhibitor. Moreover, fulvene-5 mitigated radiation-induced DNA damage in human peripheral blood mononuclear cells ex vivo. Our results provide evidence that the inactivation of NOXs protects cells from radiation-induced DNA damage and cell death. These findings suggest that NOXs inhibition may be considered as a future pharmacological target to help minimize the negative effects of radiation exposure for millions of patients each year.
Assuntos
Ciclopentanos/administração & dosagem , Dano ao DNA/genética , Proteínas de Membrana/genética , NADPH Oxidases/genética , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos da radiação , Humanos , Proteínas de Membrana/antagonistas & inibidores , NADPH Oxidase 4 , NADPH Oxidase 5 , NADPH Oxidases/antagonistas & inibidores , Cultura Primária de Células , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismoRESUMO
PURPOSE: DMS612 is a dimethane sulfonate analog with bifunctional alkylating activity and preferential cytotoxicity to human renal cell carcinoma (RCC) in the NCI-60 cell panel. This first-in-human phase I study aimed to determine dose-limiting toxicity (DLT), maximum tolerated dose (MTD), pharmacokinetics (PK), and pharmacodynamics (PD) of DMS612 administered by 10-minute intravenous infusion on days 1, 8, and 15 of an every-28-day schedule. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were eligible. Enrollment followed a 3+3 design. PKs of DMS612 and metabolites were assessed by mass spectroscopy and PD by γ-H2AX immunofluorescence. RESULTS: A total of 31 patients, including those with colorectal (11), RCC (4), cervical (2), and urothelial (1) cancers, were enrolled. Six dose levels were studied, from 1.5 mg/m(2) to 12 mg/m(2). DLTs of grade 4 neutropenia and prolonged grade 3 thrombocytopenia were observed at 12 mg/m(2). The MTD was determined to be 9 mg/m(2) with a single DLT of grade 4 thrombocytopenia in 1 of 12 patients. Two patients had a confirmed partial response at the 9 mg/m(2) dose level, in renal (1) and cervical (1) cancer. DMS612 was rapidly converted into active metabolites. γ-H2AX immunofluorescence revealed dose-dependent DNA damage in both peripheral blood lymphocytes and scalp hairs. CONCLUSIONS: The MTD of DMS12 on days 1, 8, and 15 every 28 days was 9 mg/m(2). DMS612 appears to be an alkylating agent with unique tissue specificities. Dose-dependent PD signals and two partial responses at the MTD support further evaluation of DMS612 in phase II trials.