RESUMEN
Cortical synucleinopathies, including dementia with Lewy bodies and Parkinson's disease dementia, collectively known as Lewy body dementia, are characterized by the aberrant aggregation of misfolded α-synuclein (α-syn) protein into large inclusions in cortical tissue, leading to impairments in proteostasis and synaptic connectivity and eventually resulting in neurodegeneration. Here, we show that male and female rat cortical neurons exposed to exogenous α-syn preformed fibrils accumulate large, detergent-insoluble, PS129-labeled deposits at synaptic terminals. Live-cell imaging of calcium dynamics coupled with assessment of network activity reveals that aberrant intracellular accumulation of α-syn inhibits synaptic response to glutamate through NMDARs, although deficits manifest slowly over a 7 d period. Impairments in NMDAR activity temporally correlated with increased nitric oxide synthesis and S-nitrosylation of the dendritic scaffold protein, microtubule-associated protein 1A. Inhibition of nitric oxide synthesis via the nitric oxide synthase inhibitor l-NG-nitroarginine methyl ester blocked microtubule-associated protein 1A S-nitrosylation and normalized NMDAR-dependent inward calcium transients and overall network activity. Collectively, these data suggest that loss of synaptic function in Lewy body dementia may result from synucleinopathy-evoked nitrosative stress and subsequent NMDAR dysfunction.SIGNIFICANCE STATEMENT This work shows the importance of the redox state of microtubule-associated protein 1A in the maintenance of synaptic function through regulation of NMDAR. We show that α-syn preformed fibrils promote nitric oxide synthesis, which triggers S-nitrosylation of microtubule-associated protein 1A, leading to impairment of NMDAR-dependent glutamate responses. This offers insight into the mechanism of synaptic dysfunction in Lewy body dementia.
Asunto(s)
Demencia , Enfermedad por Cuerpos de Lewy , Enfermedad de Parkinson , Sinucleinopatías , Masculino , Femenino , Animales , Ratas , alfa-Sinucleína/metabolismo , Enfermedad por Cuerpos de Lewy/metabolismo , Calcio/metabolismo , Óxido Nítrico/metabolismo , Enfermedad de Parkinson/metabolismo , Sinucleinopatías/metabolismo , Receptores de N-Metil-D-Aspartato , Glutamatos , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
Neuronal loss in Parkinson's disease (PD) is associated with impaired proteostasis and accumulation of α-syn microaggregates in dopaminergic neurons. These microaggregates promote seeding of α-synuclein (α-syn) pathology between synaptically linked neurons. However, the mechanism by which seeding is initiated is not clear. Using human pluripotent stem cell (hPSC) models of PD that allow comparison of SNCA mutant cells with isogenic controls, we find that SNCA mutant neurons accumulate α-syn deposits that cluster to multiple endomembrane compartments, specifically multivesicular bodies (MVBs) and lysosomes. We demonstrate that A53T and E46K α-syn variants bind and sequester LC3B monomers into detergent-insoluble microaggregates on the surface of late endosomes, increasing α-syn excretion via exosomes and promoting seeding of α-syn from SNCA mutant neurons to wild-type (WT) isogenic controls. Finally, we show that constitutive inactivation of LC3B promotes α-syn accumulation and seeding, while LC3B activation inhibits these events, offering mechanistic insight into the spread of synucleinopathy in PD.
Asunto(s)
Exocitosis/genética , Exosomas/metabolismo , Enfermedad de Parkinson/genética , alfa-Sinucleína/metabolismo , Diferenciación Celular , Humanos , Mutación , Enfermedad de Parkinson/patología , TransfecciónRESUMEN
Alpha-synuclein (α-syn) is a small presynaptic protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). It localizes to presynaptic terminals where it partitions between a cytosolic soluble and a lipid-bound state. Recent evidence suggests that α-syn can also associate with mitochondrial membranes where it interacts with a unique anionic phospholipid cardiolipin (CL). Here, we examine the conformation of the flexible fragments of a monomeric α-syn bound to lipid vesicles composed of anionic 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipids, of tetraoleoyl CL (TOCL) and DOPC, and of fibrils. The dynamic properties of α-syn associated with DOPA:DOPC vesicles were the most favorable for conducting three-dimensional NMR experiments, and the 13C, 15N and amide 1H chemical shifts of the flexible and disordered C-terminus of α-syn could be assigned using three-dimensional through-bond magic angle spinning NMR spectroscopy. Although the C-terminus is more dynamically constrained in fibrils and in α-syn bound to TOCL:DOPC vesicles, a direct comparison of carbon chemical shifts detected using through bond two-dimensional spectroscopy indicates that the C-terminus is flexible and unstructured in all the three samples.
Asunto(s)
alfa-SinucleínaRESUMEN
Evidence suggests that n-3 polyunsaturated fatty acids may act as activators of the Nrf2 antioxidant pathway. The antioxidant response, in turn, promotes neuronal differentiation and neurite outgrowth. Nrf2 has recently been suggested to be a cell intrinsic mediator of docosohexanoic acid (DHA) signaling. In the current study, we assessed whether DHA-mediated axodendritic development was dependent on activation of the Nrf2 pathway and whether Nrf2 protected from agrochemical-induced neuritic retraction. Expression profiling of the DHA-enriched Fat-1 mouse brain relative to wild type showed a significant enrichment of genes associated with neuronal development and neuronal projection and genes associated with the Nrf2-transcriptional pathway. Moreover, we found that primary cortical neurons treated with DHA showed a dose-dependent increase in Nrf2 transcriptional activity and Nrf2-target gene expression. DHA-mediated activation of Nrf2 promoted neurite outgrowth and inhibited oxidative stress-induced neuritic retraction evoked by exposure to agrochemicals. Finally, we provide evidence that this effect is largely dependent on induction of the Nrf2-target gene NAD(P)H: (quinone acceptor) oxidoreductase 1 (NQO1), and that silencing of either Nrf2 or Nqo1 blocks the effects of DHA on the axodendritic compartment. Collectively, these data support a role for the Nrf2-NQO1 pathway in DHA-mediated axodendritic development and protection from agrochemical exposure.
Asunto(s)
NAD(P)H Deshidrogenasa (Quinona)/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Proyección Neuronal/fisiología , Animales , Antioxidantes/farmacología , Línea Celular Tumoral , Dendritas/metabolismo , Ácidos Docosahexaenoicos/farmacología , Humanos , Ratones , Factor 2 Relacionado con NF-E2/genética , Proyección Neuronal/genética , Neuronas/metabolismo , Oxidación-Reducción , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
While mutations in the SNCA gene (α-synuclein [α-syn]) are causal in rare familial forms of Parkinson's disease (PD), the prevalence of α-syn aggregates in the cortices of sporadic disease cases emphasizes the need to understand the link between α-syn accumulation and disease pathogenesis. By employing a combination of human pluripotent stem cells (hPSCs) that harbor the SNCA-A53T mutation contrasted against isogenic controls, we evaluated the consequences of α-syn accumulation in human A9-type dopaminergic (DA) neurons (hNs). We show that the early accumulation of α-syn in SNCA-A53T hNs results in changes in gene expression consistent with the expression profile of the substantia nigra (SN) from PD patients, analyzed post mortem. Differentially expressed genes from both PD patient SN and SNCA-A53T hNs were associated with regulatory motifs transcriptionally activated by the antioxidant response pathway, particularly Nrf2 gene targets. Differentially expressed gene targets were also enriched for gene ontologies related to microtubule binding processes. We thus assessed the relationship between Nrf2-mediated gene expression and neuritic pathology in SNCA-A53T hNs. We show that SNCA-mutant hNs have deficits in neuritic length and complexity relative to isogenic controls as well as contorted axons with Tau-positive varicosities. Furthermore, we show that mutant α-syn fails to complex with protein kinase C (PKC), which, in turn, results in impaired activation of Nrf2. These neuritic defects result from impaired Nrf2 activity on antioxidant response elements (AREs) localized to a microtubule-associated protein (Map1b) gene enhancer and are rescued by forced expression of Map1b as well as by both Nrf2 overexpression and pharmaceutical activation in PD neurons.
Asunto(s)
Proteínas Asociadas a Microtúbulos/genética , Factor 2 Relacionado con NF-E2/genética , Enfermedad de Parkinson/genética , alfa-Sinucleína/genética , Animales , Elementos de Respuesta Antioxidante/genética , Axones/efectos de los fármacos , Axones/patología , Diferenciación Celular/genética , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/patología , Elementos de Facilitación Genéticos , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Mutación , Neuritas/metabolismo , Neuritas/patología , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Proteína Quinasa C/genética , Sustancia Negra/metabolismo , Sustancia Negra/patologíaRESUMEN
Neuronal loss in Parkinson's disease (PD) is associated with aberrant mitochondrial function and impaired proteostasis. Identifying the mechanisms that link these pathologies is critical to furthering our understanding of PD pathogenesis. Using human pluripotent stem cells (hPSCs) that allow comparison of cells expressing mutant SNCA (encoding α-synuclein (α-syn)) with isogenic controls, or SNCA-transgenic mice, we show that SNCA-mutant neurons display fragmented mitochondria and accumulate α-syn deposits that cluster to mitochondrial membranes in response to exposure of cardiolipin on the mitochondrial surface. Whereas exposed cardiolipin specifically binds to and facilitates refolding of α-syn fibrils, prolonged cardiolipin exposure in SNCA-mutants initiates recruitment of LC3 to the mitochondria and mitophagy. Moreover, we find that co-culture of SNCA-mutant neurons with their isogenic controls results in transmission of α-syn pathology coincident with mitochondrial pathology in control neurons. Transmission of pathology is effectively blocked using an anti-α-syn monoclonal antibody (mAb), consistent with cell-to-cell seeding of α-syn.
Asunto(s)
Cardiolipinas/farmacología , Mitocondrias/efectos de los fármacos , Membranas Mitocondriales/efectos de los fármacos , Neuronas/metabolismo , Enfermedad de Parkinson Secundaria/genética , alfa-Sinucleína/genética , Animales , Anticuerpos Monoclonales/farmacología , Comunicación Celular , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Femenino , Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Mitofagia/efectos de los fármacos , Mutación , Neuronas/efectos de los fármacos , Neuronas/patología , Enfermedad de Parkinson Secundaria/metabolismo , Enfermedad de Parkinson Secundaria/patología , Pliegue de Proteína/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , alfa-Sinucleína/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Pre-clinical data have shown that PARP inhibitors (PARPi) may increase the efficacy of radiotherapy in prostate cancer. However, it is uncertain as to whether PARPi lead to clonogenic kill when combined with radiotherapy (RT). MATERIAL AND METHODS: We tested the PARP inhibitor AZD-2281 as a radiosensitizing agent under oxic and hypoxic conditions for clonogenic survival in vitro and in vivo using the human prostate cancer cell line, 22Rv1. In addition, the effects of PARPi+RT on normal tissue were investigated using a crypt clonogenic assay. RESULTS: AZD-2281 inhibited cellular PARP activity under both oxic and hypoxic conditions. The addition of AZD-2281 radiosensitized 22Rv1 cells under oxia, acute hypoxia and chronic hypoxia in vitro. The combination of AZD-2281 with fractionated radiotherapy resulted in a significant growth delay and clonogenic kill in vivo. No increased gut toxicity was observed using this combined PARPi+radiotherapy regimen. CONCLUSIONS: This is the first preclinical study to demonstrate direct clonogenic kill in vivo by the addition of AZD-2281 to radiotherapy. As we did not observe gut toxicity, the use of PARPi in the context of prostate cancer radiotherapy warrants further investigation in clinical trials.
Asunto(s)
Antineoplásicos/farmacología , Quimioradioterapia/métodos , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Neoplasias de la Próstata/terapia , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Antineoplásicos/farmacocinética , Línea Celular Tumoral , Fraccionamiento de la Dosis de Radiación , Evaluación Preclínica de Medicamentos , Humanos , Masculino , Ratones Desnudos , Ftalazinas/farmacocinética , Piperazinas/farmacocinética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacocinética , Neoplasias de la Próstata/patología , Carga TumoralRESUMEN
PURPOSE: PTEN deletions in prostate cancer are associated with tumor aggression and poor outcome. Recent studies have implicated PTEN as a determinant of homologous recombination (HR) through defective RAD51 function. Similar to BRCA1/2-defective tumor cells, PTEN-null prostate and other cancer cells have been reported to be sensitive to PARP inhibitors (PARPi). To date, no direct comparison between PTEN and RAD51 expression in primary prostate tumors has been reported. EXPERIMENTAL DESIGN: Prostate cancer cell lines and xenografts with known PTEN status (22RV1-PTEN(+/+), DU145-PTEN(+/-), PC3-PTEN(-/-)) and H1299 and HCT116 cancer cells were used to evaluate how PTEN loss affects RAD51 expression and PARPi sensitivity. Primary prostate cancers with known PTEN status were analyzed for RAD51 expression. RESULTS: PTEN status is not associated with reduced RAD51 mRNA or protein expression in primary prostate cancers. Decreased PTEN expression did not reduce RAD51 expression or clonogenic survival following PARPi among prostate cancer cells that vary in TP53 and PTEN. PARPi sensitivity instead associated with a defect in MRE11 expression. PTEN-deficient cells had only mild PARPi sensitivity and no loss of HR or RAD51 recruitment. Clonogenic cell survival following a series of DNA damaging agents was variable: PTEN-deficient cells were sensitive to ionizing radiation, mitomycin-C, UV, H(2)O(2), and methyl methanesulfonate but not to cisplatin, camptothecin, or paclitaxel. CONCLUSIONS: These data suggest that the relationship between PTEN status and survival following DNA damage is indirect and complex. It is unlikely that PTEN status will be a direct biomarker for HR status or PARPi response in prostate cancer clinical trials.
Asunto(s)
Eliminación de Gen , Fosfohidrolasa PTEN/genética , Neoplasias de la Próstata/enzimología , Neoplasias de la Próstata/genética , Recombinasa Rad51/metabolismo , Animales , Línea Celular Tumoral , Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Expresión Génica , Silenciador del Gen , Humanos , Proteína Homóloga de MRE11 , Masculino , Proteínas de Fusión Oncogénica/genética , Fosfohidrolasa PTEN/metabolismo , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Neoplasias de la Próstata/terapia , Recombinasa Rad51/genética , Rayos Ultravioleta/efectos adversosRESUMEN
The ATM kinase is activated by chromatin modification following exogenous and endogenous DSBs or cell stress, including acute anoxia. The p53 binding protein 1 (53BP1) contains multiple ATM-consensus phosphorylation sites in its N- and C-termini and may therefore be a distal read-out of ATM function. We have examined the cellular activation of these phosphorylation sites for the first time in situ following anoxic/hypoxic stress and IR-induced exogenous DSBs. We show that multiple residues of 53BP1 are phosphorylated and that these phosphoforms form discrete nuclear foci following IR or during DNA replication as exogenous or endogenous DNA double strand breaks (DSBs), respectively. Novel data pertaining to the phosphorylation of 53BP1(Ser25)in situ supports its dependency on the ATM kinase; but this occurs independently of p53 function. We show that 53BP1(Ser25) is activated specifically in S-phase cells during anoxia in an ATM-dependent manner. Exogenous DSBs form discrete IR-induced foci whereas oxygen stress induced non-localized 53BP1(Ser25) activation. Our in vitro data are supported by irradiated xenograft studies in vivo whereby 53BP1(Ser25) phosphorylation does not occur in sub-regions positive for the hypoxia marker EF5. We propose a model whereby DSBs induce chromatin modification at sites of DNA damage which are tracked by the ATM substrates γ H2AX and 53BP1(Ser25) in a mechanism distinct from p53-mediated cell cycle arrest. Together this work indicates 53BP1(Ser25), and possibly other 53BP1 phosphoforms, as a bona fide DSB-biomarkers for surveying ongoing DNA-damage related signaling in oxic and hypoxic cells during clinical radiotherapy.
Asunto(s)
Hipoxia de la Célula , Roturas del ADN de Doble Cadena , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Western Blotting , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Humanos , Fosforilación , Radiación Ionizante , Transducción de Señal , Estadísticas no Paramétricas , Células Tumorales Cultivadas/enzimología , Células Tumorales Cultivadas/efectos de la radiación , Microambiente Tumoral , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
AKT is hyper-activated in many human cancers and promotes proliferation and cancer cell survival in response to DNA damaging agents. Ionizing radiation (IR) produces DNA double strand breaks (DSB) and activates AKT, however a direct mechanism linking intra-nuclear DSB and AKT signaling is lacking. Here we demonstrate that AKT is phosphorylated following IR in benign and malignant cells and, using colony-forming assays and in vitro rejoining assays, show that AKT promotes non-homologous end joining-mediated DSB repair and cell survival following IR. Further studies revealed that pAKT-S473, but not pAKT-T308 or total AKT, accumulates in the vicinity of IR-induced DSB and co-localizes with γH2AX and ATM-pSer1981. Based on whole-cell IR, nuclear UV microbeam, and endonuclease-induced DSB studies, we observed that pAKT-S473 is up-regulated by a DSB-induced signaling cascade, and this is dependent on the DSB sensor protein, MRE11. MRE11-dependent pAKT-S473 did not require the MRE11 endonuclease domain. The histone ubiquitin ligase RNF168 is also required for DSB-induced pAKT-S473, and DSB-induced pAKT-S473 is independent of DNA-PKcs, PI3K, and ATR. These data demonstrate that DSB activate a signaling cascade that directly promotes a PI3K-independent pathway of AKT phosphorylation that is dependent on MRE11-ATM-RNF168 signaling. Thus, these data directly link the presence of DNA breaks to AKT-mediated cell survival and support AKT as a target for cancer therapy.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral/efectos de la radiación , Supervivencia Celular , Células Cultivadas , Reparación del ADN , Proteínas de Unión al ADN/genética , Fibroblastos/citología , Fibroblastos/fisiología , Histonas/antagonistas & inhibidores , Histonas/genética , Histonas/metabolismo , Humanos , Proteína Homóloga de MRE11 , Neoplasias/genética , Neoplasias/fisiopatología , Fosfohidrolasa PTEN/genética , Fosfohidrolasa PTEN/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Radiación Ionizante , Transducción de Señal/fisiología , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
We have previously shown that the Ser15-phosphorylated p53 phosphoform, p53(Ser15), can localize at sites of ionizing radiation-induced DNA damage. In this study, we hypothesized that the non-specific DNA binding domain (NSDBD) of the p53 carboxy-terminus (C-terminus) mediates chromatin anchoring at sites of DNA damage to interact with two key mediators of the DNA damage response (DDR): ATM and 53BP1. Exogenous YFP-p53 fusion constructs expressing C-terminus deletion mutants of p53 were transfected into p53-null H1299 cells and tracked by microscopy and biochemistry to determine relative chromatin-binding pre- and postirradiation. We observed that exogenous YFP-p53(WT) and YFP-p53(Δ367-393) associated with ATM(Ser1981) and 53BP1 in the nuclear, chromatin-bound fractions after DNA damage. Of interest, YFP-p53(Δ1-299) fusion proteins, which lack transcriptional trans-activation and the Ser15-residue, bound to ATM(Ser1981) but not to 53BP1. In support of these data, we used subnuclear UV-microbeam and immunoprecipitation analyses of irradiated normal human fibroblasts (HDFs) that confirmed an interaction between endogenous p53 and ATM or 53BP1. Based on these observations, we propose a model whereby a pre-existing pool of p53 responds immediately to radiation-induced DNA damage using the C-terminus to spatially facilitate protein-protein interactions and the DDR at sites of DNA damage.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada , Línea Celular , Cromatina/metabolismo , Humanos , Fosfoproteínas/química , Fosfoproteínas/genética , Fosforilación/efectos de la radiación , Unión Proteica/efectos de la radiación , Transporte de Proteínas/efectos de la radiación , Eliminación de Secuencia , Serina/metabolismo , Transducción de Señal/efectos de la radiación , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70, and DNA ligase IV. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G(0)-G(1) and S-G(2)-M cells resulted in upregulation of Rad51 gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair-deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization.
Asunto(s)
Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias/patología , Proteínas Proto-Oncogénicas c-myc/deficiencia , Recombinación Genética , Animales , Apoptosis , Western Blotting , Ciclo Celular , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/genética , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Acute and chronic hypoxia exists within the three-dimensional microenvironment of solid tumors and drives therapy resistance, genetic instability, and metastasis. Replicating cells exposed to either severe acute hypoxia (16 hours with 0.02% O(2)) followed by reoxygenation or moderate chronic hypoxia (72 hours with 0.2% O(2)) treatments have decreased homologous recombination (HR) protein expression and function. As HR defects are synthetically lethal with poly(ADP-ribose) polymerase 1 (PARP1) inhibition, we evaluated the sensitivity of repair-defective hypoxic cells to PARP inhibition. Although PARP inhibition itself did not affect HR expression or function, we observed increased clonogenic killing in HR-deficient hypoxic cells following chemical inhibition of PARP1. This effect was partially reversible by RAD51 overexpression. PARP1(-/-) murine embryonic fibroblasts (MEF) showed a proliferative disadvantage under hypoxic gassing when compared with PARP1(+/+) MEFs. PARP-inhibited hypoxic cells accumulated γH2AX and 53BP1 foci as a consequence of altered DNA replication firing during S phase-specific cell killing. In support of this proposed mode of action, PARP inhibitor-treated xenografts displayed increased γH2AX and cleaved caspase-3 expression in RAD51-deficient hypoxic subregions in vivo, which was associated with decreased ex vivo clonogenic survival following experimental radiotherapy. This is the first report of selective cell killing of HR-defective hypoxic cells in vivo as a consequence of microenvironment-mediated "contextual synthetic lethality." As all solid tumors contain aggressive hypoxic cells, this may broaden the clinical utility of PARP and DNA repair inhibition, either alone or in combination with radiotherapy and chemotherapy, even in tumor cells lacking synthetically lethal, genetic mutations.
Asunto(s)
Línea Celular Tumoral/patología , Anaerobiosis , Animales , División Celular , Hipoxia de la Célula , Supervivencia Celular , Reparación del ADN/genética , Replicación del ADN , Fibroblastos/citología , Fibroblastos/fisiología , Células HCT116/patología , Humanos , Ratones , Ratones Desnudos , Mitosis , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , ARN Interferente Pequeño/genética , Recombinación Genética , Trasplante HeterólogoRESUMEN
PURPOSE: Rapidly metabolizing tumor cells have elevated levels of nicotinamide phosphoribosyltransferase, an enzyme involved in NAD(+) biosynthesis, which serves as an important substrate for proteins involved in DNA repair. GMX1777, which inhibits nicotinamide phosphoribosyltransferase, was evaluated in two human head and neck cancer models in combination with radiotherapy. EXPERIMENTAL DESIGN: Effects of GMX1777-mediated radiosensitization were examined via metabolic and cytotoxicity assays in vitro; mechanism of action, in vivo antitumor efficacy, and radiosensitization were also investigated. RESULTS: IC(50) values of GMX1777 for FaDu and C666-1 cells were 10 and 5 nmol/L, respectively, which interacted synergistically with radiotherapy. GMX1777 induced a rapid decline in intracellular NAD(+) followed by ATP reduction associated with significant cytotoxicity. These metabolic changes were slightly increased with the addition of radiotherapy, although poly(ADP-ribose) polymerase activity was significantly reduced when GMX1777 was combined with radiotherapy, thereby accounting for the synergistic cytotoxicity of these two modalities. Systemic GMX1777 administration with local tumor radiotherapy caused complete disappearance of FaDu and C666-1 tumors for 50 and 20 days, respectively. There was also significant reduction in tumor vascularity, particularly for the more sensitive FaDu model. [(18)F]FDG-positron emission tomography/computed tomography images showed reduction in [(18)F]FDG uptake after GMX1777 administration, showing decreased glucose metabolism in vivo. CONCLUSIONS: Our data represent the first report showing that GMX1777 plus radiotherapy is an effective therapeutic strategy for head and neck cancer, mediated via pleiotropic effects of inhibition of DNA repair and tumor angiogenesis, while sparing normal tissues. Therefore, GMX1777 combined with radiotherapy definitely warrants clinical evaluation in human head and neck cancer patients.
Asunto(s)
Guanidinas/administración & dosificación , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/radioterapia , Nicotinamida Fosforribosiltransferasa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Animales , Carcinoma de Células Escamosas/irrigación sanguínea , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/radioterapia , Terapia Combinada , Reparación del ADN/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Guanidinas/farmacología , Neoplasias de Cabeza y Cuello/irrigación sanguínea , Humanos , Ratones , Ratones SCID , NAD/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The chemo- and radioresponse of tumor cells can be determined by genetic factors (e.g., those that modify cell cycle arrest, DNA damage repair or cell death) and microenvironmental factors, such as hypoxia. Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that rapidly recognizes and binds to DNA breaks to facilitate DNA strand break repair. Pre-clinical data suggest that PARP inhibitors (PARPi) may potentiate the effects of radiotherapy and chemotherapy. However, it is unclear as to whether PARPi are effective against hypoxic cells. We therefore tested the role for a novel PARPi, ABT-888, as a radiosensitizing agent under hypoxic conditions. Using human prostate (DU-145, 22RV1) and non-small cell lung (H1299) cancer cell lines, we observed that ABT-888 inhibited both recombinant PARP activity and intracellular PARP activity (86% to 92% decrease in all 3 cells lines following 2.5 microM treatment). ABT-888 was toxic to both oxic and hypoxic cells. When ABT-888 was combined with ionizing radiation (IR), clonogenic radiation survival was decreased by 40-50% under oxic conditions. Under acute hypoxia, ABT-888 radiosensitized malignant cells to a level similar to oxic radiosensitivity. To our knowledge, this is the first study to demonstrate that inhibition of PARP activity can sensitize hypoxic cancer cells and the combination of IR-PARPi has the potential to improve the therapeutic ratio of radiotherapy.
Asunto(s)
Bencimidazoles/farmacología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Reparación del ADN/efectos de los fármacos , Neoplasias Pulmonares/terapia , Neoplasias de la Próstata/tratamiento farmacológico , Fármacos Sensibilizantes a Radiaciones/farmacología , Análisis de Varianza , Apoptosis , Western Blotting , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/radioterapia , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Hipoxia , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/radioterapia , MasculinoRESUMEN
New molecular cancer treatment strategies aim to reconstitute wild-type p53 (WTp53) function in mutant p53 (MTp53)-expressing tumors as a means of resensitizing cells to chemotherapy or radiotherapy. The success of this approach may depend on whether MTp53 proteins are acting in a dominant-negative or independent gain-of-function mode. Herein, we describe an isogenic, temperature-sensitive p53 model (p53(A138V)) in p53-null human H1299 lung cancer cells in which WTp53 can be selectively coexpressed with a temperature-sensitive MTp53 allele (A138V) during initial DNA damage and subsequent DNA repair. Cells expressing MTp53 alone or coexpressing induced WTp53 and MTp53 were tested for p53 transcription, G(1) and G(2) cell cycle checkpoints, apoptosis, and long-term clonogenic survival following DNA damage. Transient transfection of WTp53 into H1299 cells, or shift-down of H1299-p53(A138V) stable transfectants to 32 degrees C to induce WTp53, led to increased p21(WAF1) expression and G(1) and G(2) arrests following DNA damage but did not increase BAX expression or apoptosis. In contrast, both transient and stable expression of the p53(A138V) mutant in p53-null H1299 cells (e.g. testing gain-of-function) at 37 degrees C blocked p21(WAF1) induction following DNA damage. Cell death was secondary to mitotic catastrophe and/or tumor cell senescence. Overexpression of WTp53 did not resensitize resistant MTp53-expressing cells to ionizing radiation, cisplatinum, or mitomycin C. Our results suggest that human MTp53 proteins can lead to resistant phenotypes independent of WTp53-mediated transcription and checkpoint control. This should be considered when using p53 as a prognostic factor and therapeutic target.
Asunto(s)
Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Tolerancia a Radiación/genética , Proteína p53 Supresora de Tumor/genética , Antibióticos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Senescencia Celular , Cisplatino/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN/efectos de los fármacos , Daño del ADN/efectos de la radiación , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Fase G1/efectos de los fármacos , Fase G1/efectos de la radiación , Fase G2/efectos de los fármacos , Fase G2/efectos de la radiación , Rayos gamma , Humanos , Immunoblotting , Inmunoprecipitación , Mitomicina/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Radiación Ionizante , Fármacos Sensibilizantes a Radiaciones/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Transcripción Genética/efectos de la radiación , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/efectos de la radiación , Ensayo de Tumor de Célula Madre , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Despite a clear link between ataxia-telangiectasia mutated (ATM)-dependent phosphorylation of p53 and cell cycle checkpoint control, the intracellular biology and subcellular localization of p53 phosphoforms during the initial sensing of DNA damage is poorly understood. Using G0-G1 confluent primary human diploid fibroblast cultures, we show that endogenous p53, phosphorylated at Ser15 (p53Ser15), accumulates as discrete, dose-dependent and chromatin-bound foci within 30 minutes following induction of DNA breaks or DNA base damage. This biologically distinct subpool of p53Ser15 is ATM dependent and resistant to 26S-proteasomal degradation. p53Ser15 colocalizes and coimmunoprecipitates with gamma-H2AX with kinetics similar to that of biochemical DNA double-strand break (DNA-dsb) rejoining. Subnuclear microbeam irradiation studies confirm p53Ser15 is recruited to sites of DNA damage containing gamma-H2AX, ATM(Ser1981), and DNA-PKcs(Thr2609) in vivo. Furthermore, studies using isogenic human and murine cells, which express Ser15 or Ser18 phosphomutant proteins, respectively, show defective nuclear foci formation, decreased induction of p21WAF, decreased gamma-H2AX association, and altered DNA-dsb kinetics following DNA damage. Our results suggest a unique biology for this p53 phosphoform in the initial steps of DNA damage signaling and implicates ATM-p53 chromatin-based interactions as mediators of cell cycle checkpoint control and DNA repair to prevent carcinogenesis.
Asunto(s)
Daño del ADN , ADN/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ácido Anhídrido Hidrolasas , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Reparación del ADN , Enzimas Reparadoras del ADN/metabolismo , Proteína Quinasa Activada por ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Fibroblastos/metabolismo , Fibroblastos/efectos de la radiación , Células HCT116 , Histonas/metabolismo , Humanos , Inmunoprecipitación , Ratones , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal , Proteínas Supresoras de Tumor/metabolismoRESUMEN
PURPOSE: To test the hypotheses that deliberate elevation of PaCO(2) increases cerebral tissue oxygen tension (PBrO(2)) by augmenting PaO(2) and regional cerebral blood flow (rCBF). METHODS: Anesthetized rats were exposed to increasing levels of inspired oxygen (O(2)) or carbon dioxide (CO(2); 5%, 10% and 15%, n = 6). Mean arterial blood pressure (MAP), PBrO(2) and rCBF were measured continuously. Blood gas analysis and hemoglobin concentrations were determined for each change in inspired gas concentration. Data are presented as mean +/- standard deviation with P < 0.05 taken to be significant. RESULTS: The PBrO(2) increased in proportion to arterial oxygenation (PaO(2)) when the percentage of inspired O(2) was increased. Proportional increases in PaCO(2) (48.7 +/- 4.9, 72.3 +/- 6.0 and 95.3 +/- 15.4 mmHg), PaO(2) (172.2 +/- 33.1, 191.7 +/- 42.5 and 216.0 +/- 41.8 mmHg), and PBrO(2) (29.1 +/- 9.2, 49.4 +/- 19.5 and 60.5 +/- 23.0 mmHg) were observed when inspired CO(2) concentrations were increased from 0% to 5%, 10% and 15%, respectively, while arterial pH decreased (P < 0.05 for each). Exposure to CO(2) increased rCBF from 1.04 +/- 0.67 to a peak value of 1.49 +/- 0.45 (P < 0.05). Following removal of exogenous CO(2), arterial blood gas values returned to baseline while rCBF and PBrO(2) remained elevated for over 30 min. The hypercapnia induced increase in PBrO(2) was threefold higher than that resulting from a comparable increase in PaO(2) achieved by increasing the inspired O(2) concentration (34.9 +/- 14.5 vs 11.4 +/- 5.0 mmHg, P < 0.05). CONCLUSION: These data support the hypothesis that the combined effect of increased CBF, PaO(2) and reduced pH collectively contribute to augmenting cerebral PBrO(2) during hypercapnia.