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1.
Redox Biol ; 24: 101206, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31039479

RESUMO

We tested whether novel CYP11A1-derived vitamin D3- and lumisterol-hydroxyderivatives, including 1,25(OH)2D3, 20(OH)D3, 1,20(OH)2D3, 20,23(OH)2D3, 1,20,23(OH)3D3, lumisterol, 20(OH)L3, 22(OH)L3, 20,22(OH)2L3, and 24(OH)L3, can protect against UVB-induced damage in human epidermal keratinocytes. Cells were treated with above compounds for 24 h, then subjected to UVB irradiation at UVB doses of 25, 50, 75, or 200 mJ/cm2, and then examined for oxidant formation, proliferation, DNA damage, and the expression of genes at the mRNA and protein levels. Oxidant formation and proliferation were determined by the DCFA-DA and MTS assays, respectively. DNA damage was assessed using the comet assay. Expression of antioxidative genes was evaluated by real-time RT-PCR analysis. Nuclear expression of CPD, phospho-p53, and Nrf2 as well as its target proteins including HO-1, CAT, and MnSOD, were assayed by immunofluorescence and western blotting. Treatment of cells with the above compounds at concentrations of 1 or 100 nM showed a dose-dependent reduction in oxidant formation. At 100 nM they inhibited the proliferation of cultured keratinocytes. When keratinocytes were irradiated with 50-200 mJ/cm2 of UVB they also protected against DNA damage, and/or induced DNA repair by enhancing the repair of 6-4PP and attenuating CPD levels and the tail moment of comets. Treatment with test compounds increased expression of Nrf2-target genes involved in the antioxidant response including GR, HO-1, CAT, SOD1, and SOD2, with increased protein expression for HO-1, CAT, and MnSOD. The treatment also stimulated the phosphorylation of p53 at Ser-15, increased its concentration in the nucleus and enhanced Nrf2 translocation into the nucleus. In conclusion, pretreatment of keratinocytes with 1,25(OH)2D3 or CYP11A1-derived vitamin D3- or lumisterol hydroxy-derivatives, protected them against UVB-induced damage via activation of the Nrf2-dependent antioxidant response and p53-phosphorylation, as well as by the induction of the DNA repair system. Thus, the new vitamin D3 and lumisterol hydroxy-derivatives represent promising anti-photodamaging agents.

2.
Adv Protein Chem Struct Biol ; 115: 247-295, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30798934

RESUMO

Malignant melanoma of the skin is the leading cause of death from skin cancer and ranks fifth in cancer incidence among all cancers in the United States. While melanoma mortality has remained steady for the past several decades, melanoma incidence has been increasing, particularly among fair-skinned individuals. According to the American Cancer Society, nearly 10,000 people in the United States will die from melanoma this year. Individuals with dark skin complexion are protected damage generated by UV-light due to the high content of UV-blocking melanin pigment in their epidermis as well as better capacity for melanocytes to cope with UV damage. There is now ample evidence that suggests that the melanocortin 1 receptor (MC1R) is a major melanoma risk factor. Inherited loss-of-function mutations in MC1R are common in melanoma-prone persons, correlating with a less melanized skin complexion and poorer recovery from mutagenic photodamage. We and others are interested in the MC1R signaling pathway in melanocytes, its mechanisms of enhancing genomic stability and pharmacologic opportunities to reduce melanoma risk based on those insights. In this chapter, we review melanoma risk factors, the MC1R signaling pathway, and the relationship between MC1R signaling and DNA repair.


Assuntos
AMP Cíclico/metabolismo , Instabilidade Genômica , Melanócitos/metabolismo , Melanoma/genética , Melanoma/prevenção & controle , Animais , Humanos , Melanoma/metabolismo , Melanoma/patologia
3.
J Biol Chem ; 293(49): 19025-19037, 2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30327428

RESUMO

Blunted melanocortin 1 receptor (MC1R) signaling promotes melanocyte genomic instability in part by attenuating cAMP-mediated DNA repair responses, particularly nucleotide excision repair (NER), which recognizes and clears mutagenic photodamage. cAMP-enhanced NER is mediated by interactions between the ataxia telangiectasia-mutated and Rad3-related (ATR) and xeroderma pigmentosum complementation group A (XPA) proteins. We now report a critical role for sirtuin 1 (SIRT1) in regulating ATR-mediated phosphorylation of XPA. SIRT1 deacetylates XPA at residues Lys-63, Lys-67, and Lys-215 to promote interactions with ATR. Mutant XPA containing acetylation mimetics at residues Lys-63, Lys-67, and Lys-215 exhibit blunted UV-dependent ATR-XPA interactions even in the presence of cAMP signals. ATR-mediated phosphorylation of XPA on Ser-196 enhances cAMP-mediated optimization of NER and is promoted by SIRT1-mediated deacetylation of XPA on Lys-63, Lys-67, and Lys-215. Interference with ATR-mediated XPA phosphorylation at Ser-196 by persistent acetylation of XPA at Lys-63, Lys-67, and Lys-215 delays repair of UV-induced DNA damage and attenuates cAMP-enhanced NER. Our study identifies a regulatory ATR-SIRT1-XPA axis in cAMP-mediated regulation melanocyte genomic stability, involving SIRT1-mediated deacetylation (Lys-63, Lys-67, and Lys-215) and ATR-dependent phosphorylation (Ser-196) post-translational modifications of the core NER factor XPA.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Reparo do DNA/fisiologia , Sirtuína 1/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Acetilação , Linhagem Celular Tumoral , AMP Cíclico/metabolismo , Humanos , Lisina/química , Melanócitos/efeitos da radiação , Fosforilação , Processamento de Proteína Pós-Traducional , Serina/química , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/química
4.
Sci Rep ; 7(1): 11708, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28916831

RESUMO

Using primary melanocytes and HEK293 cells, we found that cAMP signaling accelerates repair of bi- and mono-functional platinum-induced DNA damage. Elevating cAMP signaling either by the agonistic MC1R ligand melanocyte stimulating hormone (MSH) or by pharmacologic cAMP induction by forskolin enhanced clearance of intrastrand cisplatin-adducts in melanocytes or MC1R-transfected HEK293 cells. MC1R antagonists human beta-defensin 3 and agouti signaling protein blocked MSH- but not forskolin-mediated enhancement of platinum-induced DNA damage. cAMP-enhanced repair of cisplatin-induced DNA damage was dependent on PKA-mediated phosphorylation of ATR on S435 which promoted ATR's interaction with the key NER factor xeroderma pigmentosum A (XPA) and facilitated recruitment of an XPA-ATR-pS435 complex to sites of cisplatin DNA damage. Moreover, we developed an oligonucleotide retrieval immunoprecipitation (ORiP) assay using a novel platinated-DNA substrate to establish kinetics of ATR-pS435 and XPA's associations with cisplatin-damaged DNA. Expression of a non-phosphorylatable ATR-S435A construct or deletion of A kinase-anchoring protein 12 (AKAP12) impeded platinum adduct clearance and prevented cAMP-mediated enhancement of ATR and XPA's associations with cisplatin-damaged DNA, indicating that ATR phosphorylation at S435 is necessary for cAMP-enhanced repair of platinum-induced damage and protection against cisplatin-induced mutagenesis. These data implicate cAMP signaling as a critical regulator of genomic stability against platinum-induced mutagenesis.


Assuntos
AMP Cíclico/metabolismo , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Melanocortinas/fisiologia , Mutagênese/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Instabilidade Genômica/efeitos dos fármacos , Células HEK293 , Humanos , Compostos de Platina/toxicidade , Transdução de Sinais/fisiologia
5.
Sci Rep ; 7(1): 1274, 2017 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-28455491

RESUMO

Ultraviolet light (UV) is an inducer of reactive oxygen species (ROS) as well as 6-4-photoproducts and cyclobutane pyrimidine dimers (CPD) in the skin, which further cause damage to the skin cells. Irradiation of cultured human melanocytes with UVB stimulated ROS production, which was reduced in cells treated with melatonin or its metabolites: 6-hydroxymelatonin (6-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), and 5-methoxytryptamine (5-MT). Melatonin and its derivatives also stimulated the expression of NRF2 (nuclear factor erythroid 2 [NF-E2]-related factor 2) and its target enzymes and proteins that play an important role in cell protection from different damaging factors including UVB. Silencing of NRF2 using siRNA diminished the protective effects of melatonin, while the membrane melatonin receptors (MT1 or MT2) did not change the activities of either melatonin or its derivatives. Melatonin and its metabolites enhanced the DNA repair in melanocytes exposed to UVB and stimulated expression of p53 phosphorylated at Ser-15. In conclusion, melatonin and its metabolites protect melanocytes from UVB-induced DNA damage and oxidative stress through activation of NRF2-dependent pathways; these actions are independent of an effect on the classic membrane melatonin receptors. Thus, melatonin and its derivatives can serve as excellent protectors of melanocytes against UVB-induced pathology.


Assuntos
Melanócitos/fisiologia , Melanócitos/efeitos da radiação , Melatonina/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Protetores contra Radiação/metabolismo , Raios Ultravioleta , Células Cultivadas , Reparo do DNA/efeitos dos fármacos , Humanos , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/toxicidade
6.
Exp Dermatol ; 26(7): 577-584, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28094871

RESUMO

Loss-of-function melanocortin 1 receptor (MC1R) polymorphisms are common in UV-sensitive fair-skinned individuals and are associated with blunted cAMP second messenger signalling and higher lifetime risk of melanoma because of diminished ability of melanocytes to cope with UV damage. cAMP signalling positions melanocytes to resist UV injury by upregulating synthesis of UV-blocking eumelanin pigment and by enhancing the repair of UV-induced DNA damage. cAMP enhances melanocyte nucleotide excision repair (NER), the genome maintenance pathway responsible for the removal of mutagenic UV photolesions, through cAMP-activated protein kinase (protein kinase A)-mediated phosphorylation of the ataxia telangiectasia-mutated and Rad3-related (ATR) protein on the S435 residue. We investigated the interdependence of cAMP-mediated melanin upregulation and cAMP-enhanced DNA repair in primary human melanocytes and a melanoma cell line. We observed that the ATR-dependent molecular pathway linking cAMP signalling to the NER pathway is independent of MITF activation. Similarly, cAMP-mediated upregulation of pigment synthesis is independent of ATR, suggesting that the key molecular events driving MC1R-mediated enhancement of genome maintenance (eg PKA-mediated phosphorylation of ATR) and MC1R-induced pigment induction (eg MITF activation) are distinct.


Assuntos
AMP Cíclico/metabolismo , Reparo do DNA , Melanócitos/citologia , Receptor Tipo 1 de Melanocortina/metabolismo , Pigmentação da Pele , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Dano ao DNA , Humanos , Levodopa/química , Melaninas/química , Mutagênese , Nucleotídeos/química , Fosforilação , RNA Interferente Pequeno/metabolismo , Receptor Tipo 1 de Melanocortina/genética , Serina/química , Serina/genética , Transdução de Sinais , Raios Ultravioleta , Regulação para Cima
7.
Photochem Photobiol ; 93(1): 245-258, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27645605

RESUMO

Melanoma is the deadliest form of skin cancer because of its propensity to spread beyond the primary site of disease and because it resists many forms of treatment. Incidence of melanoma has been increasing for decades. Although ultraviolet radiation (UV) has been identified as the most important environmental causative factor for melanoma development, UV-protective strategies have had limited efficacy in melanoma prevention. UV mutational burden correlates with melanoma development and tumor progression, underscoring the importance of UV in melanomagenesis. However, besides amount of UV exposure, melanocyte UV mutational load is influenced by the robustness of nucleotide excision repair, the genome maintenance pathway charged with removing UV photoproducts before they cause permanent mutations in the genome. In this review, we highlight the importance of the melanocortin hormonal signaling axis on regulating efficiency of nucleotide excision repair in melanocytes. By understanding the molecular mechanisms by which nucleotide excision repair can be increased, it may be possible to prevent many cases of melanoma by reducing UV mutational burden over time.


Assuntos
Reparo do DNA , Melanocortinas/metabolismo , Melanócitos/metabolismo , Dímeros de Pirimidina/metabolismo , Transdução de Sinais , Raios Ultravioleta/efeitos adversos , AMP Cíclico/metabolismo , Humanos , Melanoma/epidemiologia , Receptor Tipo 1 de Melanocortina/agonistas , Receptor Tipo 1 de Melanocortina/antagonistas & inibidores , Receptor Tipo 1 de Melanocortina/metabolismo , Estados Unidos/epidemiologia , Xeroderma Pigmentoso/etiologia
8.
Nucleic Acids Res ; 44(22): 10711-10726, 2016 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-27683220

RESUMO

Loss-of-function in melanocortin 1 receptor (MC1R), a GS protein-coupled receptor that regulates signal transduction through cAMP and protein kinase A (PKA) in melanocytes, is a major inherited melanoma risk factor. Herein, we report a novel cAMP-mediated response for sensing and responding to UV-induced DNA damage regulated by A-kinase-anchoring protein 12 (AKAP12). AKAP12 is identified as a necessary participant in PKA-mediated phosphorylation of ataxia telangiectasia mutated and Rad3-related (ATR) at S435, a post-translational event required for cAMP-enhanced nucleotide excision repair (NER). Moreover, UV exposure promotes ATR-directed phosphorylation of AKAP12 at S732, which promotes nuclear translocation of AKAP12-ATR-pS435. This complex subsequently recruits XPA to UV DNA damage and enhances 5' strand incision. Preventing AKAP12's interaction with PKA or with ATR abrogates ATR-pS435 accumulation, delays recruitment of XPA to UV-damaged DNA, impairs NER and increases UV-induced mutagenesis. Our results define a critical role for AKAP12 as an UV-inducible scaffold for PKA-mediated ATR phosphorylation, and identify a repair complex consisting of AKAP12-ATR-pS435-XPA at photodamage, which is essential for cAMP-enhanced NER.


Assuntos
Proteínas de Ancoragem à Quinase A/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Dano ao DNA , Reparo do DNA , Células HEK293 , Humanos , Cinética , Mutagênese , Fosforilação , Transporte Proteico , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
9.
J Invest Dermatol ; 135(12): 3086-3095, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26168232

RESUMO

The melanocortin 1 receptor (MC1R), a GS-coupled receptor that signals through cAMP and protein kinase A (PKA), regulates pigmentation, adaptive tanning, and melanoma resistance. MC1R-cAMP signaling promotes PKA-mediated phosphorylation of ataxia telangiectasia and rad3-related (ATR) at Ser435 (ATR-pS435), a modification that enhances nucleotide excision repair (NER) by facilitating recruitment of the XPA protein to sites of UV-induced DNA damage. High-throughput methods were developed to quantify ATR-pS435, measure XPA-photodamage interactions, and assess NER function. We report that melanocyte-stimulating hormone (α-MSH) or ACTH induce ATR-pS435, enhance XPA's association with UV-damaged DNA and optimize melanocytic NER. In contrast, MC1R antagonists agouti signaling protein (ASIP) or human ß-defensin 3 (HBD3) interfere with ATR-pS435 generation, impair the XPA-DNA interaction, and reduce DNA repair. Although ASIP and HBD3 each blocked α-MSH-mediated induction of the signaling pathway, only ASIP depleted basal ATR-pS435. Our findings confirm that ASIP diminishes agonist-independent MC1R basal signaling whereas HBD3 is a neutral MC1R antagonist that blocks activation by melanocortins. Furthermore, our data suggest that ATR-pS435 may be a useful biomarker for the DNA repair-deficient MC1R phenotype.


Assuntos
Reparo do DNA , Melanócitos/metabolismo , Receptor Tipo 1 de Melanocortina/fisiologia , Proteína Agouti Sinalizadora/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Biomarcadores , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , DNA/metabolismo , Humanos , Fosforilação , Serina/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , beta-Defensinas/farmacologia
10.
Naunyn Schmiedebergs Arch Pharmacol ; 388(2): 199-206, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25017017

RESUMO

The NME1 gene represents the prototypical metastasis suppressor, whose expression inhibits cell motility and metastasis without impact on primary tumor growth in a number of different human cancers. This report outlines our recent efforts to define the molecular mechanisms through which NME1 both suppresses cell motility and promotes genomic integrity in the setting of human melanoma. Forced NME1 expression in a variety of melanoma-derived cell lines was shown to induce dynamic changes in cell morphology and reorganization of the actin cytoskeleton, with formation of a network of thick stress fibers and assembly of fibronectin fibrils at large focal adhesions. Moreover, NME1 expression results in adhesion reprogramming through an impact on integrin repertoire and focal adhesion dynamics. Having previously demonstrated that NME1 expression promotes repair of DNA damage induced by ultraviolet radiation (UVR) in both yeast and mammalian cells, probably via the nucleotide excision repair pathway, we have more recently demonstrated that NME1 is rapidly recruited to double-strand breaks. This preliminary result represents the first evidence of direct interactions between NME1 and DNA in the context of DNA repair and has set the stage for current efforts to probe its functional interactions with double-strand break repair pathways. Discussed herein are molecular models to explain the interactions of NME1 with such diverse cellular functions as cell motility and DNA repair, potentially through its nucleoside diphosphate kinase and 3'-5' exonuclease activities.


Assuntos
Melanoma , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neoplasias Cutâneas , Actinas/metabolismo , Animais , Movimento Celular , Reparo do DNA , Adesões Focais , Instabilidade Genômica , Humanos , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia
11.
Mol Cell ; 54(6): 999-1011, 2014 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-24950377

RESUMO

The melanocortin 1 receptor (MC1R), which signals through cAMP, is a melanocytic transmembrane receptor involved in pigmentation, adaptive tanning, and melanoma resistance. We report MC1R-mediated or pharmacologically-induced cAMP signaling promotes nucleotide excision repair (NER) in a cAMP-dependent protein kinase A (PKA)-dependent manner. PKA directly phosphorylates ataxia telangiectasia and Rad3-related protein (ATR) at Ser435, which actively recruits the key NER protein xeroderma pigmentosum complementation group A (XPA) to sites of nuclear UV photodamage, accelerating clearance of UV-induced photolesions and reducing mutagenesis. Loss of Ser435 within ATR prevents PKA-mediated ATR phosphorylation, disrupts ATR-XPA binding, delays recruitment of XPA to UV-damaged DNA, and elevates UV-induced mutagenesis. This study mechanistically links cAMP-PKA signaling to NER and illustrates potential benefits of cAMP pharmacological rescue to reduce UV mutagenesis in MC1R-defective, melanoma-susceptible individuals.


Assuntos
Dano ao DNA , Receptor Tipo 1 de Melanocortina/genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia/química , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese/efeitos da radiação , Fosforilação/efeitos da radiação , Pigmentação/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/efeitos da radiação , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/genética , Transdução de Sinais/efeitos da radiação , Raios Ultravioleta , Proteína de Xeroderma Pigmentoso Grupo A/genética
13.
J Pineal Res ; 57(1): 90-102, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24867336

RESUMO

We investigated the protective effects of melatonin and its metabolites: 6-hydroxymelatonin (6-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), and 5-methoxytryptamine (5-MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm2) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50-60%) when UVB-exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose-dependent increase in viability of UVB-irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot-blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB-induced pyrimidine photoproducts (6-4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser-15 but not at Ser-46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6-OHM, AFMK, 5-MT, which are endogenously produced in keratinocytes, protect these cells against UVB-induced oxidative stress and DNA damage.


Assuntos
Queratinócitos/efeitos dos fármacos , Queratinócitos/efeitos da radiação , Melatonina/farmacologia , Raios Ultravioleta , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Humanos , Cinuramina/farmacologia , Melatonina/análogos & derivados , Serotonina/análogos & derivados , Serotonina/farmacologia
14.
Methods Mol Biol ; 935: 227-43, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23150372

RESUMO

Mitochondrial dysfunction and genomic instability are associated with a number of retinal pathologies including age-related macular degeneration, diabetic retinopathy, and glaucoma. Consequences of mitochondrial dysfunction within cells include elevation of the rate of ROS production due to damage of electron transport chain proteins, mitochondrial DNA (mtDNA) damage, and loss of metabolic capacity. Here we introduce the quantitative polymerase chain reaction assay (QPCR) and extracellular flux assay (XF) as powerful techniques to study mitochondrial behavior. The QPCR technique is a gene-specific assay developed to analyze the DNA damage repair response in mitochondrial and nuclear genomes. QPCR has proved particularly valuable for the measurement of oxidative-induced mtDNA damage and kinetics of mtDNA repair. To assess the functional consequence of mitochondrial oxidative damage, real-time changes in cellular bioenergetics of cell monolayers can be measured with a Seahorse Biosciences XF24 analyzer. The advantages and limitations of these procedures will be discussed and detailed methodologies provided with particular emphasis on retinal oxidative stress.


Assuntos
Dano ao DNA , DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Retina/citologia , Animais , Técnicas de Cultura de Células/métodos , DNA Mitocondrial/isolamento & purificação , Humanos , Mitocôndrias/patologia , Estresse Oxidativo , Oxigênio/metabolismo , Retina/metabolismo , Retina/patologia
15.
Clin Exp Metastasis ; 30(1): 25-36, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22699362

RESUMO

Cutaneous malignant melanoma is the most lethal form of skin cancer, with 5-year survival rates of <5 % for patients presenting with metastatic disease. Mechanisms underlying metastatic spread of UVR-induced melanoma are not well understood, in part due to a paucity of animal models that accurately recapitulate the disease in its advanced forms. We have employed a transgenic mouse strain harboring a tandem deletion of the nm23-m1 and nm23-m2 genes to assess the combined contribution of these genes to suppression of melanoma metastasis. Crossing of the nm23-h1/nm23-h2 knockout in hemizygous-null form ([m1m2](+/-)) to a transgenic mouse strain (hepatocyte growth factor/scatter factor-overexpressing, or HGF(+) strain) vulnerable to poorly-metastatic, UVR-induced melanomas resulted in UVR-induced melanomas with high metastatic potential. Metastasis to draining lymph nodes was seen in almost all cases of back skin melanomas, while aggressive metastasis to lung, thoracic cavity, liver and bone also occurred. Interestingly, no differences were observed in the invasive characteristics of primary melanomas of HGF(+) and HGF(+) × [m1m2](+/-) strains, with both exhibiting invasion into the dermis and subcutis, indicating factors other than simple invasive activity were responsible for metastasis of HGF(+) × [m1m2](+/-) melanomas. Stable cell lines were established from the primary and metastatic melanoma lesions from these mice, with HGF(+) × [m1m2](+/-) lines exhibiting increased single cell migration and genomic instability. These studies demonstrate for the first time in vivo a potent metastasis suppressor activity of NM23 in UVR-induced melanoma, and have provided new tools for identifying molecular mechanisms that underlie melanoma metastasis.


Assuntos
Modelos Animais de Doenças , Instabilidade Genômica , Melanoma/etiologia , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Neoplasias Cutâneas/etiologia , Raios Ultravioleta/efeitos adversos , Animais , Movimento Celular , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Fator de Crescimento de Hepatócito/genética , Humanos , Hipoxantina Fosforribosiltransferase/genética , Masculino , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação/genética , Neoplasias Cutâneas/secundário , Células Tumorais Cultivadas , Cicatrização
16.
Mol Aspects Med ; 33(4): 399-417, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22510306

RESUMO

The retina resides in an environment that is primed for the generation of reactive oxygen species (ROS) and resultant oxidative damage. The retina is one of the highest oxygen-consuming tissues in the human body. The highest oxygen levels are found in the choroid, but this falls dramatically across the outermost retina, creating a large gradient of oxygen towards the retina and inner segments of the photoreceptors which contain high levels of polyunsaturated fatty acids. This micro-environment together with abundant photosensitizers, visible light exposure and a high energy demand supports a highly oxidative milieu. However, oxidative damage is normally minimized by the presence of a range of antioxidant and efficient repair systems. Unfortunately, as we age oxidative damage increases, antioxidant capacity decreases and the efficiency of reparative systems become impaired. The result is retinal dysfunction and cell loss leading to visual impairment. It appears that these age-related oxidative changes are a hallmark of early age-related macular degeneration (AMD) which, in combination with hereditary susceptibility and other retinal modifiers, can progress to the pathology and visual morbidity associated with advanced AMD. This review reassesses the consequences of oxidative stress in AMD and strategies for preventing or reversing oxidative damage in retinal tissues.


Assuntos
Degeneração Macular/etiologia , Degeneração Macular/metabolismo , Estresse Oxidativo , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Humanos , Degeneração Macular/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Retina/patologia
17.
Cancer Res ; 72(1): 133-43, 2012 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-22080566

RESUMO

Reduced expression of the metastasis suppressor NM23-H1 is associated with aggressive forms of multiple cancers. Here, we establish that NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3'-5' exonuclease and nucleoside diphosphate kinase, are novel participants in the cellular response to UV radiation (UVR)-induced DNA damage. NM23-H1 deficiency compromised the kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision repair of (6-4) photoproducts in vitro. Kinase activity of NM23-H1 was critical for rapid repair of both polychromatic UVB/UVA-induced (290-400 nm) and UVC-induced (254 nm) DNA damage, whereas its 3'-5' exonuclease activity was dominant in the suppression of UVR-induced mutagenesis. Consistent with its role in DNA repair, NM23-H1 rapidly translocated to sites of UVR-induced (6-4) photoproduct DNA damage in the nucleus. In addition, transgenic mice hemizygous-null for nm23-m1 and nm23-m2 exhibited UVR-induced melanoma and follicular infundibular cyst formation, and tumor-associated melanocytes displayed invasion into adjacent dermis, consistent with loss of invasion-suppressing activity of NM23 in vivo. Taken together, our data show a critical role for NM23 isoforms in limiting mutagenesis and suppressing UVR-induced melanomagenesis.


Assuntos
Dano ao DNA , Melanoma Experimental/prevenção & controle , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Neoplasias Induzidas por Radiação/prevenção & controle , Raios Ultravioleta , Animais , Linhagem Celular Tumoral , Hipoxantina Fosforribosiltransferase/genética , Melanoma Experimental/etiologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Nucleosídeo NM23 Difosfato Quinases/genética
18.
Naunyn Schmiedebergs Arch Pharmacol ; 384(4-5): 433-8, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21448569

RESUMO

nm23-h1 was the first metastasis suppressor gene to be identified in humans, with early studies demonstrating its ability to inhibit the metastatic potential of breast carcinoma and melanoma cell lines. This report outlines recent findings from our laboratory indicating that the metastasis suppressor function of NM23-H1 in human melanoma involves a spectrum of molecular mechanisms. Analysis of NM23-H1-dependent profiles of gene expression in human melanoma cell lines has identified a host of target genes that appear to mediate suppression of directional motility. Of particular interest is a subset of motility-suppressing genes whose regulation by NM23-H1 is independent of its known kinase and 3'-5' exonuclease activities. In parallel, we have recently observed that NM23-H1 expression appears to be required for genomic stability and for optimal repair of DNA damage produced by ultraviolet radiation and other agents. Thus, NM23-H1 might oppose not only the motile and invasive characteristics of metastatic cells but also the acquisition of mutations that drive malignant progression to the metastatic phenotype itself.


Assuntos
Melanoma/patologia , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Quimiocinas/genética , Feminino , Humanos , Melanoma/enzimologia , Melanoma/genética , Camundongos , Nucleosídeo NM23 Difosfato Quinases/genética , Invasividade Neoplásica , Metástase Neoplásica
19.
Ophthalmic Res ; 44(3): 179-90, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20829642

RESUMO

Mitochondria are critical for ocular function as they represent the major source of a cell's supply of energy and play an important role in cell differentiation and survival. Mitochondrial dysfunction can occur as a result of inherited mitochondrial mutations (e.g. Leber's hereditary optic neuropathy and chronic progressive external ophthalmoplegia) or stochastic oxidative damage which leads to cumulative mitochondrial damage and is an important factor in age-related disorders (e.g. age-related macular degeneration, cataract and diabetic retinopathy). Mitochondrial DNA (mtDNA) instability is an important factor in mitochondrial impairment culminating in age-related changes and pathology, and in all regions of the eye mtDNA damage is increased as a consequence of aging and age-related disease. It is now apparent that the mitochondrial genome is a weak link in the defenses of ocular cells since it is susceptible to oxidative damage and it lacks some of the systems that protect the nuclear genome, such as nucleotide excision repair. Accumulation of mitochondrial mutations leads to cellular dysfunction and increased susceptibility to adverse events which contribute to the pathogenesis of numerous sporadic and chronic disorders in the eye.


Assuntos
Envelhecimento/fisiologia , DNA Mitocondrial/genética , Oftalmopatias Hereditárias/genética , Mitocôndrias/fisiologia , Mutação , Animais , Humanos , Espécies Reativas de Oxigênio
20.
Mol Cell Biochem ; 329(1-2): 161-5, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19381784

RESUMO

nm23-h1 is a well-documented metastasis suppressor gene whose mechanism(s) of action have yet to be fully elucidated. The purpose of this report is to discuss recent advances in investigating the potential role of a novel 3'-5' exonuclease activity identified recently in our laboratory, a biochemical function associated, in general, with DNA repair and replication. We have employed a site-directed mutagenesis approach to demonstrate that the 3'-5' exonuclease activity of NM23-H1 is required for its metastasis suppressor function. Consistent with a role in DNA repair, we also observe that the single yeast NM23 homolog (YNK1) is required for the maintenance of genomic integrity and normal kinetics of DNA repair in response to exposure to ultraviolet radiation. These results and their implications for understanding the molecular mechanisms underlying NM23-H1 functions in cancer are discussed.


Assuntos
Reparo do DNA , Exonucleases/genética , Genes Supressores de Tumor , Nucleosídeo NM23 Difosfato Quinases/genética , Metástase Neoplásica/genética , Linhagem Celular Tumoral , Exonucleases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Mutagênese Sítio-Dirigida , Mutação/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Metástase Neoplásica/prevenção & controle
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