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1.
BMC Bioinformatics ; 20(1): 725, 2019 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-31852428

RESUMEN

BACKGROUND: Macrophages show versatile functions in innate immunity, infectious diseases, and progression of cancers and cardiovascular diseases. These versatile functions of macrophages are conducted by different macrophage phenotypes classified as classically activated macrophages and alternatively activated macrophages due to different stimuli in the complex in vivo cytokine environment. Dissecting the regulation of macrophage activations will have a significant impact on disease progression and therapeutic strategy. Mathematical modeling of macrophage activation can improve the understanding of this biological process through quantitative analysis and provide guidance to facilitate future experimental design. However, few results have been reported for a complete model of macrophage activation patterns. RESULTS: We globally searched and reviewed literature for macrophage activation from PubMed databases and screened the published experimental results. Temporal in vitro macrophage cytokine expression profiles from published results were selected to establish Boolean network models for macrophage activation patterns in response to three different stimuli. A combination of modeling methods including clustering, binarization, linear programming (LP), Boolean function determination, and semi-tensor product was applied to establish Boolean networks to quantify three macrophage activation patterns. The structure of the networks was confirmed based on protein-protein-interaction databases, pathway databases, and published experimental results. Computational predictions of the network evolution were compared against real experimental results to validate the effectiveness of the Boolean network models. CONCLUSION: Three macrophage activation core evolution maps were established based on the Boolean networks using Matlab. Cytokine signatures of macrophage activation patterns were identified, providing a possible determination of macrophage activations using extracellular cytokine measurements.


Asunto(s)
Citocinas/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Modelos Teóricos
2.
BMC Cancer ; 17(1): 864, 2017 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-29254481

RESUMEN

BACKGROUND: The global incidence of melanoma has been increasing faster than any other form of cancer. New therapies offer exciting prospects for improved survival, but the development of resistance is a major problem and there remains a need for additional effective melanoma therapy. Platinum compounds, such as cisplatin, are the most effective chemotherapeutics for a number of major cancers, but are ineffective on metastatic melanoma. They cause monofunctional adducts and intrastrand crosslinks that are repaired by nucleotide excision repair, as well as the more toxic interstrand crosslinks that are repaired by a combination of nuclease activity and homologous recombination. METHODS: We investigated the mechanism of melanoma resistance to cisplatin using a panel of melanoma and control cell lines. Cisplatin-induced changes in levels of the key homologous recombination protein RAD51 and compensatory changes in translesion synthesis DNA polymerases were identified by western blotting and qRT-PCR. Flow cytometry, immunofluorescence and western blotting were used to compare the cell cycle and DNA damage response and the induction of apoptosis in cisplatin-treated melanoma and control cells. Ectopic expression of a tagged form of RAD51 and siRNA knockdown of translesion synthesis DNA polymerase zeta were used to investigate the mechanism that allowed cisplatin-treated melanoma cells to continue to replicate. RESULTS: We have identified and characterised a novel DNA damage response mechanism in melanoma. Instead of increasing levels of RAD51 on encountering cisplatin-induced interstrand crosslinks during replication, melanoma cells shut down RAD51 synthesis and instead boost levels of translesion synthesis DNA polymerase zeta to allow replication to proceed. This response also resulted in synthetic lethality to the PARP inhibitor olaparib. CONCLUSIONS: This unusual DNA damage response may be a more appropriate strategy for an aggressive and rapidly growing tumour like melanoma that enables it to better survive chemotherapy, but also results in increased sensitivity of cultured melanoma cells to the PARP inhibitor olaparib.


Asunto(s)
Recombinación Homóloga/genética , Melanoma/tratamiento farmacológico , Recombinasa Rad51/genética , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/administración & dosificación , Daño del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Recombinación Homóloga/efectos de los fármacos , Humanos , Melanoma/genética , Melanoma/patología , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Inhibidores de Poli(ADP-Ribosa) Polimerasas/administración & dosificación
3.
Physiol Genomics ; 48(5): 345-60, 2016 05.
Artículo en Inglés | MEDLINE | ID: mdl-26884460

RESUMEN

Dynamic, epigenetic mechanisms can regulate macrophage phenotypes following exposure to different stimulating conditions and environments. However, temporal patterns of microRNAs (miRNAs or miRs) across multiple macrophage polarization phenotypes have not been defined. We determined miRNA expression in bone marrow-derived murine macrophages over multiple time points (0.5, 1, 3, 24 h) following exposure to cytokines and/or LPS. We hypothesized that dynamic changes in miRNAs regulate macrophage phenotypes. Changes in macrophage polarization markers were detected as early as 0.5 and as late as 24 h; however, robust responses for most markers occurred within 3 h. In parallel, many polarization-specific miRNAs were also changed by 3 h and expressed divergent patterns between M1 and M2a conditions, with increased expression in M1 (miR-155, 199a-3p, 214-3p, 455-3p, and 125a) or M2a (miR-511 and 449a). Specifically, miR-125a-5p exhibited divergent patterns: increased at 12-24 h in M1 macrophages and decreasing trend in M2a. VEGF in the culture media of macrophages was dependent upon the polarization state, with greatly diminished VEGF in M2a compared with M1 macrophage culture media despite similar VEGF in cell lysates. Inhibition of miR-125a-5p in media-only controls (MO) and M1 macrophages greatly increased expression and secretion of soluble VEGF receptor-1 (sVEGFR1) leading to diminished VEGF in the culture media, partially converting MO and M1 into an M2a phenotype. Thus, the divergent expression patterns of polarization-specific miRNAs led to the identification and demonstrated the regulation of a specific macrophage polarization phenotype, sVEGFR1 by inhibition of miR-125a-5p.


Asunto(s)
Macrófagos/metabolismo , MicroARNs/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Animales , Células Cultivadas , Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Fenotipo , Factor A de Crecimiento Endotelial Vascular/genética
4.
Am J Pathol ; 184(4): 1167-1184, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24525152

RESUMEN

Monocyte/macrophage polarization in skeletal muscle regeneration is ill defined. We used CD11b-diphtheria toxin receptor transgenic mice to transiently deplete monocytes/macrophages at multiple stages before and after muscle injury induced by cardiotoxin. Fat accumulation within regenerated muscle was maximal when ablation occurred at the same time as cardiotoxin-induced injury. Early ablation (day 1 after cardiotoxin) resulted in the smallest regenerated myofiber size together with increased residual necrotic myofibers and fat accumulation. However, muscle regeneration after late (day 4) ablation was similar to controls. Levels of inflammatory cells in injured muscle following early ablation and associated with impaired muscle regeneration were determined by flow cytometry. Delayed, but exaggerated, monocyte [CD11b(+)(CD90/B220/CD49b/NK1.1/Ly6G)(-)(F4/80/I-Ab/CD11c)(-)Ly6C(+/-)] accumulation occurred; interestingly, Ly6C(+) and Ly6C(-) monocytes were present concurrently in ablated animals and control mice. In addition to monocytes, proinflammatory, Ly6C(+) macrophage accumulation following early ablation was delayed compared to controls. In both groups, CD11b(+)F4/80(+) cells exhibited minimal expression of the M2 markers CD206 and CD301. Nevertheless, early ablation delayed and decreased the transient accumulation of CD11b(+)F4/80(+)Ly6C(-)CD301(-) macrophages; in control animals, the later tissue accumulation of these cells appeared to correspond to that of anti-inflammatory macrophages, determined by cytokine production and arginase activity. In summary, impairments in muscle regeneration were associated with exaggerated monocyte recruitment and reduced Ly6C(-) macrophages; the switch of macrophage/monocyte subsets is critical to muscle regeneration.


Asunto(s)
Macrófagos/citología , Músculo Esquelético/patología , Músculo Esquelético/fisiología , Animales , Quimiotaxis de Leucocito , Citometría de Flujo , Ratones , Ratones Transgénicos , Músculo Esquelético/lesiones , Fenotipo , Regeneración
5.
Bioorg Med Chem Lett ; 25(19): 4104-8, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26321360

RESUMEN

A high throughput screen allowed the identification of N-hydroxyimide inhibitors of ERCC1-XPF endonuclease activity with micromolar potency, but they showed undesirable selectivity profiles against FEN-1. A scaffold hop to a hydroxypyrimidinone template gave compounds with similar potency but allowed selectivity to be switched in favour of ERCC1-XPF over FEN-1. Further exploration of the structure-activity relationships around this chemotype gave sub-micromolar inhibitors with >10-fold selectivity for ERCC1-XPF over FEN-1.


Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Endonucleasas/antagonistas & inhibidores , Imidas/farmacología , Pirimidinonas/farmacología , Reparación del ADN , Relación Dosis-Respuesta a Droga , Endonucleasas de ADN Solapado/antagonistas & inhibidores , Células Hep G2 , Humanos , Imidas/química , Estructura Molecular , Pirimidinonas/química , Relación Estructura-Actividad
6.
Bioorg Med Chem Lett ; 25(19): 4097-103, 2015 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-26318993

RESUMEN

Catechol-based inhibitors of ERCC1-XPF endonuclease activity were identified from a high-throughput screen. Exploration of the structure-activity relationships within this series yielded compound 13, which displayed an ERCC1-XPF IC50 of 0.6 µM, high selectivity against FEN-1 and DNase I and activity in nucleotide excision repair, cisplatin enhancement and γH2AX assays in A375 melanoma cells. Screening of fragments as potential alternatives to the catechol group revealed that 3-hydroxypyridones are able to inhibit ERCC1-XPF with high ligand efficiency, and elaboration of the hit gave compounds 36 and 37 which showed promising ERCC1-XPF IC50 values of <10 µM.


Asunto(s)
Catecoles/farmacología , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Endonucleasas/antagonistas & inhibidores , Piridonas/farmacología , Catecoles/química , Línea Celular Tumoral , Desoxirribonucleasa I/antagonistas & inhibidores , Desoxirribonucleasa I/metabolismo , Relación Dosis-Respuesta a Droga , Endonucleasas de ADN Solapado/antagonistas & inhibidores , Humanos , Estructura Molecular , Piridonas/química , Relación Estructura-Actividad
7.
Gynecol Oncol ; 132(2): 506-12, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24333356

RESUMEN

DNA mismatch repair (MMR) deficiency is associated with increased risk of developing several types of cancer and is the most common cause of hereditary ovarian cancer after BRCA1 and BRCA2 mutations. While there has been extensive investigation of MMR deficiency in colorectal cancer, MMR in ovarian cancer is relatively under-investigated. This review summarizes the mechanism of MMR, the ways in which MMR deficiency can promote carcinogenesis in general and then assesses the available studies regarding MMR deficiency in ovarian cancers with specific emphasis on implications for disease incidence and therapy. The incidence of germline MMR gene mutations in ovarian cancer is only 2% but other mechanisms of gene inactivation mean that loss of expression of one of the seven main genes (MSH2, MSH3, MSH6, MLH1, MLH3, PMS1 and PMS2) occurs in up to 29% of cases. Both mutational and expression data suggest that MMR deficiency is more common in non-serous ovarian cancer. Some studies suggest an improved survival for patients with MMR deficiency compared to historical controls but these do not account for the preponderance of non-serous tumors. A number of in vitro studies have suggested that MMR deficiency is a cause of platinum resistance. To date this has not been categorically demonstrated in the clinic. Larger studies that account for stage of presentation and immunohistochemical subtype are required to assess the effect of MMR deficiency on survival and chemosensitivity. Investigation of MMR related synthetic lethality in colorectal cancer has identified dihydrofolate reductase, DNA polymerase ß and DNA polymerase γ and PTEN-induced putative kinase 1 as synthetic lethal to certain MMR defects by causing accumulation of oxidative DNA damage. These synthetic lethal targets require tested and others should be sought within the context of MMR deficient ovarian cancer in an attempt to provide novel therapeutic strategies for these patients.


Asunto(s)
Reparación de la Incompatibilidad de ADN , Neoplasias Ováricas/genética , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología
8.
Nucleic Acids Res ; 40(20): 9990-10004, 2012 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-22941649

RESUMEN

The ERCC1-XPF complex is a structure-specific endonuclease essential for the repair of DNA damage by the nucleotide excision repair pathway. It is also involved in other key cellular processes, including DNA interstrand crosslink (ICL) repair and DNA double-strand break (DSB) repair. New evidence has recently emerged, increasing our understanding of its requirement in these additional roles. In this review, we focus on the protein-protein and protein-DNA interactions made by the ERCC1 and XPF proteins and discuss how these coordinate ERCC1-XPF in its various roles. In a number of different cancers, high expression of ERCC1 has been linked to a poor response to platinum-based chemotherapy. We discuss prospects for the development of DNA repair inhibitors that target the activity, stability or protein interactions of the ERCC1-XPF complex as a novel therapeutic strategy to overcome chemoresistance.


Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Resistencia a Antineoplásicos , Endonucleasas/antagonistas & inhibidores , Neoplasias/tratamiento farmacológico , Reparación del ADN , Trastornos por Deficiencias en la Reparación del ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Descubrimiento de Drogas , Endonucleasas/química , Endonucleasas/genética , Endonucleasas/metabolismo , Humanos , Mutación , Estructura Terciaria de Proteína , Homeostasis del Telómero
9.
Hum Mol Genet ; 20(11): 2131-43, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21378394

RESUMEN

The instability of (CTG)•(CAG) repeats can cause >15 diseases including myotonic dystrophy, DM1. Instability can arise during DNA replication, repair or recombination, where sealing of nicks by DNA ligase I (LIGI) is a final step. The role of LIGI in CTG/CAG instability was determined using in vitro and in vivo approaches. Cell extracts from a human (46BR) harbouring a deficient LIGI (∼3% normal activity) were used to replicate CTG/CAG repeats; and DM1 mice with >300 CTG repeats were crossed with mice harbouring the 46BR LigI. In mice, the defective LigI reduced the frequency of CTG expansions and increased CTG contraction frequencies on female transmissions. Neither male transmissions nor somatic CTG instability was affected by the 46BR LigI - indicating a post-female germline segregation event. Replication-mediated instability was affected by the 46BR LIGI in a manner that depended upon the location of Okazaki fragment initiation relative to the repeat tract; on certain templates, the expansion bias was unaltered by the mutant LIGI, similar to paternal transmissions and somatic tissues; however, a replication fork-shift reduced expansions and increased contractions, similar to maternal transmissions. The presence of contractions in oocytes suggests that the DM1 replication profile specific to pre-meiotic oogenesis replication of maternal alleles is distinct from that occurring in other tissues and, when mediated by the mutant LigI, is predisposed to CTG contractions. Thus, unlike other DNA metabolizing enzymes studied to date, LigI has a highly specific role in CTG repeat maintenance in the maternal germline, involved in mediating CTG expansions and in the avoidance of maternal CTG contractions.


Asunto(s)
ADN Ligasas/metabolismo , Replicación del ADN , Inestabilidad Genómica , Expansión de Repetición de Trinucleótido/genética , Alelos , Animales , Línea Celular , ADN/genética , ADN Ligasa (ATP) , ADN Ligasas/genética , Análisis Mutacional de ADN , Femenino , Homocigoto , Humanos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Distrofia Miotónica/genética , Oocitos/metabolismo , Fosforilación
10.
Histopathology ; 62(2): 343-50, 2013 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-23020778

RESUMEN

AIMS: Elevated expression of DNA repair and replication genes has been reported in thick, non-fixed primary melanomas that subsequently went on to metastasize, when compared to non-recurrent primary tumours. This increased expression could contribute to the extreme resistance shown by melanoma to DNA-damaging chemotherapeutics. We have investigated the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in melanoma. METHODS AND RESULTS: We used a tissue microarray containing samples from all stages of melanomagenesis to investigate the hypothesis that levels of key DNA repair and replication proteins are prognostic biomarkers in a larger, more representative and readily available set of fixed primary melanomas. High expression of topoisomerase IIα (TOP2A), that relieves torsional stress during DNA replication, and XRCC5 (Ku80), required for DNA double-strand break repair, were associated with significantly worse survival. CONCLUSIONS: Two (XRCC5 and TOP2A) of seven DNA repair and replication proteins studied were prognostic for melanoma.


Asunto(s)
Antígenos de Neoplasias/metabolismo , ADN Helicasas/metabolismo , Reparación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Melanoma/secundario , Neoplasias Cutáneas/patología , Biomarcadores de Tumor/metabolismo , Progresión de la Enfermedad , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Autoantígeno Ku , Ganglios Linfáticos/patología , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/mortalidad , Proteínas de Unión a Poli-ADP-Ribosa , Pronóstico , Modelos de Riesgos Proporcionales , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/mortalidad , Tasa de Supervivencia , Análisis de Matrices Tisulares , Reino Unido/epidemiología
11.
J Neurosci ; 31(35): 12543-53, 2011 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-21880916

RESUMEN

Age-related cognitive decline and neurodegenerative diseases are a growing challenge for our societies with their aging populations. Accumulation of DNA damage has been proposed to contribute to these impairments, but direct proof that DNA damage results in impaired neuronal plasticity and memory is lacking. Here we take advantage of Ercc1(Δ/-) mutant mice, which are impaired in DNA nucleotide excision repair, interstrand crosslink repair, and double-strand break repair. We show that these mice exhibit an age-dependent decrease in neuronal plasticity and progressive neuronal pathology, suggestive of neurodegenerative processes. A similar phenotype is observed in mice where the mutation is restricted to excitatory forebrain neurons. Moreover, these neuron-specific mutants develop a learning impairment. Together, these results suggest a causal relationship between unrepaired, accumulating DNA damage, and age-dependent cognitive decline and neurodegeneration. Hence, accumulated DNA damage could therefore be an important factor in the onset and progression of age-related cognitive decline and neurodegenerative diseases.


Asunto(s)
Envejecimiento , Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/genética , Trastornos por Deficiencias en la Reparación del ADN/complicaciones , Degeneración Nerviosa/etiología , Degeneración Nerviosa/genética , Factor de Transcripción Activador 3/metabolismo , Factores de Edad , Análisis de Varianza , Animales , Caspasa 3/metabolismo , Trastornos del Conocimiento/metabolismo , Trastornos por Deficiencias en la Reparación del ADN/genética , Proteínas de Unión al ADN/deficiencia , Modelos Animales de Enfermedad , Estimulación Eléctrica , Endonucleasas/deficiencia , Miedo/psicología , Regulación de la Expresión Génica/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Hipocampo/patología , Hipocampo/fisiopatología , Técnicas In Vitro , Potenciación a Largo Plazo/genética , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Plasticidad Neuronal/genética , Proteína p53 Supresora de Tumor/metabolismo
12.
Physiol Genomics ; 44(21): 1042-51, 2012 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-22968638

RESUMEN

MicroRNAs (miRNAs) regulate many biological processes including muscle development. However, little is known regarding miRNA regulation of muscle regeneration. Murine tibialis anterior muscle was evaluated after cardiotoxin-induced injury and used for global miRNA expression analysis. From day 1 through day 21 following injury, 298 miRNAs were significantly changed at least at one time point, including 86 miRNAs that were altered >10-fold compared with uninjured skeletal muscle. Temporal miRNA expression patterns included inflammation-related miRNAs (miR-223 and -147) that increased immediately after injury; this pattern contrasted to that of mature muscle-specific miRNAs (miR-1, -133a, and -499) that abruptly decreased following injury followed by upregulation in later regenerative events. Another cluster of miRNAs were transiently increased in the early days of muscle regeneration including miR-351, a miRNA that was also transiently expressed during myogenic progenitor cell (MPC) differentiation in vitro. Based on computational predictions, further studies demonstrated that E2f3 was a target of miR-351 in myoblasts. Moreover, knockdown of miR-351 expression inhibited MPC proliferation and promoted apoptosis during MPC differentiation, whereas miR-351 overexpression protected MPC from apoptosis during differentiation. Collectively, these observations suggest that miR-351 is involved in both the maintenance of MPC proliferation and the transition into differentiated myotubes. Thus, a novel, time-dependent sequence of molecular events during muscle regeneration has been identified; miR-351 inhibits E2f3 expression, a key regulator of cell cycle progression and proliferation, and promotes MPC proliferation and protects early differentiating MPC from apoptosis, important events in the hostile tissue environment after acute muscle injury.


Asunto(s)
Diferenciación Celular , Proliferación Celular , MicroARNs/metabolismo , Músculo Esquelético/fisiología , Células Madre/citología , Animales , Supervivencia Celular , Masculino , Ratones , MicroARNs/genética , Músculo Esquelético/citología , Regeneración , Células Madre/metabolismo , Regulación hacia Arriba
13.
DNA Repair (Amst) ; 8(5): 664-71, 2009 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-19264557

RESUMEN

Topical application of thymidine dinucleotides (pTpT) provides some protection against the effects of UV on the skin, however, many details of the protective mechanism have yet to be elucidated. We have used mice with an epidermis-specific knockout for the nucleotide excision repair gene, Ercc1, to investigate the mechanisms of protection. pTpT offered no protection against the pronounced UV-induced short-term erythema and skin thickening responses that are characteristic of DNA repair-deficient skin. It also had no effect on UV-induced apoptosis in Ercc1-deficient cultured keratinocytes. However, in these short-term experiments in both skin and keratinocyte culture pTpT did cause a slight reduction in proliferation. pTpT application during a chronic UV irradiation protocol provided some protection from UVB-induced skin carcinogenesis in epidermis-specific Ercc1 knockout mice. The median tumour free survival time was increased in the pTpT-treated group and treated animals had fewer tumours. In addition, pTpT-treated animals developed fewer large inwardly growing skin lesions than untreated animals. Furthermore, the proliferation response was reduced in chronically irradiated, non-lesional pTpT-treated skin. We conclude that cancer protection by pTpT in our mice is not modulated by an upregulation of DNA repair, as protection appears to be independent of a functional nucleotide excision repair pathway. We hypothesise instead that protection by pTpT is due to a reduction in epidermal proliferation.


Asunto(s)
Reparación del ADN/genética , Proteínas de Unión al ADN/fisiología , Endonucleasas/fisiología , Neoplasias Inducidas por Radiación/prevención & control , Neoplasias Cutáneas/prevención & control , Timidina/administración & dosificación , Rayos Ultravioleta/efectos adversos , Animales , Apoptosis/efectos de la radiación , Western Blotting , Proliferación Celular/efectos de los fármacos , Proliferación Celular/efectos de la radiación , Células Cultivadas , Daño del ADN , Células Epidérmicas , Epidermis/efectos de los fármacos , Epidermis/efectos de la radiación , Eritema/metabolismo , Eritema/patología , Eritema/prevención & control , Femenino , Técnicas para Inmunoenzimas , Integrasas/metabolismo , Queratinocitos/citología , Queratinocitos/efectos de los fármacos , Queratinocitos/efectos de la radiación , Masculino , Ratones , Ratones Noqueados , Neoplasias Inducidas por Radiación/metabolismo , Neoplasias Inducidas por Radiación/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Tasa de Supervivencia , Irradiación Corporal Total
14.
Cancers (Basel) ; 12(6)2020 Jun 09.
Artículo en Inglés | MEDLINE | ID: mdl-32526907

RESUMEN

High grade serous ovarian cancer (HGSOC) is a major cause of female cancer mortality. The approval of poly (ADP-ribose) polymerase (PARP) inhibitors for clinical use has greatly improved treatment options for patients with homologous recombination repair (HRR)-deficient HGSOC, although the development of PARP inhibitor resistance in some patients is revealing limitations to outcome. A proportion of patients with HRR-proficient cancers also benefit from PARP inhibitor therapy. Our aim is to compare mechanisms of resistance to the PARP inhibitor olaparib in these two main molecular categories of HGSOC and investigate a way to overcome resistance that we considered particularly suited to a cancer like HGSOC, where there is a very high incidence of TP53 gene mutation, making HGSOC cells heavily reliant on the G2 checkpoint for repair of DNA damage and survival. We identified alterations in multiple factors involved in resistance to PARP inhibition in both HRR-proficient and -deficient cancers. The most frequent change was a major reduction in levels of poly (ADP-ribose) glycohydrolase (PARG), which would be expected to preserve a residual PARP1-initiated DNA damage response to DNA single-strand breaks. Other changes seen would be expected to boost levels of HRR of DNA double-strand breaks. Growth of all olaparib-resistant clones isolated could be controlled by WEE1 kinase inhibitor AZD1775, which inactivates the G2 checkpoint. Our work suggests that use of the WEE1 kinase inhibitor could be a realistic therapeutic option for patients that develop resistance to olaparib.

15.
DNA Repair (Amst) ; 7(2): 281-91, 2008 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-18221731

RESUMEN

Transcription-coupled repair of endogenous DNA damage appears crucial for the maintenance of the central and peripheral nervous systems. Ercc1 is essential for nucleotide excision repair and is also involved in recombination repair and the repair of interstrand cross-links. We have investigated the neurological phenotype of Ercc1-deficient mice where the liver dysfunction has been corrected by an Ercc1 transgene controlled by a liver-specific promoter. We observed poor coordination, ataxia and loss of visual acuity, but saw no evidence of the anticipated histopathological neurodegeneration, or of abnormal neuromuscular junctions. Instead we observed uraemic encephalopathy, a brain disease resulting from kidney failure. This diagnosis was supported by histopathological signs of kidney disease, as well as proteinuria. When we examined archival sections from neural-specific Ercc1 knockout mice, which showed the same reduced growth and died at the same age as the liver-corrected Ercc1 knockouts, we found no evidence of kidney pathology or encephalopathy. Thus, while some aspects of the Ercc1-deficient phenotype are indicative of functional neurodegeneration, we obtained no structural evidence for this. The structural changes observed in the brains of liver-corrected Ercc1 knockouts appear to be a secondary consequence of kidney failure arising from Ercc1 deficiency.


Asunto(s)
Encefalopatías Metabólicas/etiología , Reparación del ADN/genética , Proteínas de Unión al ADN/deficiencia , Endonucleasas/deficiencia , Unión Neuromuscular/patología , Fenotipo , Insuficiencia Renal/etiología , Animales , Encefalopatías Metabólicas/patología , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Inmunohistoquímica , Riñón/patología , Ratones , Proteinuria , Desempeño Psicomotor , Células de Purkinje/patología , Insuficiencia Renal/complicaciones
16.
J Biomed Biotechnol ; 2009: 901079, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19478961

RESUMEN

Amphotropic pseudotyped retroviral vectors have typically been used to infect target cells without prior concentration. Although this can yield high rates of infection, higher rates may be needed where highly efficient coinfection of two or more vectors is needed. In this investigation we used amphotropic retroviral vectors produced by the Plat-A cell line and studied coinfection rates using green and red fluorescent proteins (EGFP and dsRed2). Target cells were primary human fibroblasts (PHF) and 3T3 cells. Unconcentrated vector preparations produced a coinfection rate of approximately 4% (defined as cells that are both red and green as a percentage of all cells infected). Optimized spinoculation, comprising centrifugation at 1200 g for 2 hours at 15 degrees C, increased the coinfection rate to approximately 10%. Concentration by centrifugation at 10,000 g or by flocculation using Polybrene increased the coinfection rate to approximately 25%. Combining the two processes, concentration by Polybrene flocculation and optimized spinoculation, increased the coinfection rate to 35% (3T3) or >50% (PHF). Improved coinfection should be valuable in protocols that require high transduction by combinations of two or more retroviral vectors.


Asunto(s)
Vectores Genéticos/genética , Retroviridae/clasificación , Retroviridae/fisiología , Células 3T3 , Animales , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/virología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Luminiscentes/metabolismo , Ratones , Retroviridae/genética , Proteína Fluorescente Roja
17.
DNA Repair (Amst) ; 6(3): 304-16, 2007 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-17126084

RESUMEN

The ERCC1/XPF complex is responsible for incision at the 5' side of the lesion during nucleotide excision repair and is also involved in homologous recombination and interstrand cross-link repair. The aim of the current study was to set up a better model for examination of Ercc1 deficiency in the murine liver and to determine the DNA lesions responsible for the premature polyploidy observed. We used the Cre/lox system with an adenovirus carrying Cre recombinase to conditionally induce Ercc1 deficiency in murine hepatocytes in vitro. Increased levels of apoptosis were apparent in our Ercc1-deficient cultures, both spontaneously and after UV irradiation and oxidative DNA damage. Increased apoptosis was also observed in simple Ercc1-deficient livers and the time course of the development of polyploidy was characterised. Livers from simple Ercc1 knockout mice contained mitochondria with disrupted outer membranes. Lipid accumulation was observed in older Ercc1-deficient hepatocyte cultures and in young Ercc1-deficient and wild-type livers. Lipids disappeared from the wild-type livers with age, but persisted in Ercc1-deficient livers, suggesting that a reduced ability to repair oxidative DNA damage and a malfunction of oxidative pathways could be responsible for the Ercc1-deficient liver phenotype. Real-time RT-PCR was used to determine differences in expression of cell cycle regulation and survival genes between Ercc1-deficient and control livers. Higher mRNA levels of Igfbp2, a possible marker for polyploidy, and p21 were detected in Ercc1-deficient livers. The pro-apoptotic factor, Bax, showed increased levels of mRNA expression in young Ercc1-deficient livers. However, no elevation in the levels of reactive oxygen species, or of malondialdehyde DNA adducts, a product of oxidative DNA damage, were found in Ercc1-deficient liver and no elevated levels of genes involved in the oxidative damage response were seen.


Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/genética , Endonucleasas/genética , Hepatocitos/metabolismo , Hígado/metabolismo , Poliploidía , Animales , Apoptosis/genética , Células Cultivadas , Citoplasma/metabolismo , Aductos de ADN/metabolismo , Daño del ADN/efectos de la radiación , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/metabolismo , Endonucleasas/deficiencia , Endonucleasas/metabolismo , Glucosa Oxidasa/administración & dosificación , Hepatocitos/ultraestructura , Integrasas/metabolismo , Hígado/citología , Malondialdehído/metabolismo , Lípidos de la Membrana/metabolismo , Ratones , Ratones Noqueados , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Rayos Ultravioleta
18.
Mol Hum Reprod ; 14(1): 1-8, 2008 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-18175790

RESUMEN

In Europe up to one in four couples experience difficulty conceiving and in half of these cases the problem has been attributed to sub or infertility in the male partner. The development of assisted reproductive technologies (ART) such as in vitro fertilization and intra-cytoplasmic spermatozoa injection has allowed some such couples to achieve a pregnancy. Concerns have been raised over the increasing use of ART not least because of the discovery of elevated levels of DNA damage in sperm from subfertile men. The impact of damaged DNA originating in the male germ line is poorly understood, but is thought to contribute to early pregnancy loss (recurrent miscarriage), placental problems and have a long-term impact on the health of the offspring. DNA repair is essential for meiotic recombination and correction of DNA damage in germ cells and proteins involved in all the major repair pathways are expressed in the testis. In this review, we will consider evidence that the production of sperm containing damaged DNA can be the result of suboptimal DNA repair and/or a mild environmental insult, such as heat stress, and how studies in mice may give us insight into the origins and consequences of DNA damage in human sperm.


Asunto(s)
Reparación del ADN , Trastornos de Estrés por Calor/fisiopatología , Infertilidad Masculina/genética , Animales , Daño del ADN , Femenino , Calor , Humanos , Infertilidad Masculina/fisiopatología , Masculino , Embarazo
19.
Mol Cell Biol ; 23(24): 9245-50, 2003 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-14645534

RESUMEN

In mammals, the three classical ras genes encode four highly homologous proteins, N-Ras, H-Ras, and the isoforms K-Ras 4A and 4B. Previous studies have shown that K-ras is essential for mouse development and that while K-ras 4A and 4B are expressed during development, K-ras 4A expression is regulated temporally and spatially and occurs in adult kidney, intestine, stomach, and liver. In the present study, the pattern of K-ras 4A expression was examined in a wide range of wild-type adult mouse tissues, and gene targeting was used to generate K-ras 4A-deficient mice to examine its role in development. It was found that K-ras 4A is also expressed in uterus, lung, pancreas, salivary glands, seminal vesicles, bone marrow cells, and cecum, where it was the major K-Ras isoform expressed. Mating between K-ras(tmDelta4A/+) mice produced viable K-ras(tmDelta4A/tmDelta4A) offspring with the expected Mendelian ratios of inheritance, and these mice expressed the K-ras 4B splice variant only. K-ras(tmDelta4A/tmDelta4A) mice were fertile and showed no histopathological abnormalities on inbred (129/Ola) or crossbred (129/Ola x C57BL/6) genetic backgrounds. The results demonstrate that K-Ras 4A, like H- and N-Ras, is dispensable for normal mouse development, at least in the presence of functional K-Ras 4B.


Asunto(s)
Genes ras , Empalme Alternativo , Animales , Secuencia de Bases , ADN/genética , Femenino , Fertilidad/genética , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Isoformas de Proteínas/genética , Distribución Tisular
20.
Oncotarget ; 8(33): 55246-55264, 2017 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-28903417

RESUMEN

DNA repair pathways present in all cells serve to preserve genome stability, but in cancer cells they also act reduce the efficacy of chemotherapy. The endonuclease ERCC1-XPF has an important role in the repair of DNA damage caused by a variety of chemotherapeutic agents and there has been intense interest in the use of ERCC1 as a predictive marker of therapeutic response in non-small cell lung carcinoma, squamous cell carcinoma and ovarian cancer. We have previously validated ERCC1 as a therapeutic target in melanoma, but all small molecule ERCC1-XPF inhibitors reported to date have lacked sufficient potency and specificity for clinical use. In an alternative approach to prevent the repair activity of ERCC1-XPF, we investigated the mechanism of ERCC1 ubiquitination and found that the key region was the C-terminal (HhH)2 domain which heterodimerizes with XPF. This ERCC1 region was modified by non-conventional lysine-independent, but proteasome-dependent polyubiquitination, involving Lys33 of ubiquitin and a linear ubiquitin chain. XPF was not polyubiquitinated and its expression was dependent on presence of ERCC1, but not vice versa. To our surprise we found that ERCC1 can also homodimerize through its C-terminal (HhH)2 domain. We exploited the ability of a peptide containing this C-terminal domain to destabilise both endogenous ERCC1 and XPF in human melanoma cells and fibroblasts, resulting in reductions of up to 85% in nucleotide excision repair and near two-fold increased sensitivity to DNA damaging agents. We suggest that the ERCC1 (HhH)2 domain could be used in an alternative strategy to treat cancer.

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