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5.
Redox Biol ; 24: 101206, 2019 06.
Article in English | MEDLINE | ID: mdl-31039479

ABSTRACT

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.


Subject(s)
Cholecalciferol/pharmacology , Ergosterol/pharmacology , Keratinocytes/drug effects , Keratinocytes/metabolism , Keratinocytes/radiation effects , NF-E2-Related Factor 2/metabolism , Tumor Suppressor Protein p53/metabolism , Ultraviolet Rays/adverse effects , Antioxidants/metabolism , Cells, Cultured , Cholecalciferol/analogs & derivatives , Cholecalciferol/chemistry , DNA Damage , Ergosterol/chemistry , Gene Expression Profiling , Humans , Molecular Structure , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Oxidative Stress/radiation effects , Protective Agents/chemistry , Protective Agents/pharmacology , Signal Transduction
6.
Adv Protein Chem Struct Biol ; 115: 247-295, 2019.
Article in English | MEDLINE | ID: mdl-30798934

ABSTRACT

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.


Subject(s)
Cyclic AMP/metabolism , Genomic Instability , Melanocytes/metabolism , Melanoma/genetics , Melanoma/prevention & control , Animals , Humans , Melanoma/metabolism , Melanoma/pathology
7.
J Biol Chem ; 293(49): 19025-19037, 2018 12 07.
Article in English | MEDLINE | ID: mdl-30327428

ABSTRACT

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.


Subject(s)
Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Repair/physiology , Sirtuin 1/metabolism , Xeroderma Pigmentosum Group A Protein/metabolism , Acetylation , Cell Line, Tumor , Cyclic AMP/metabolism , Humans , Lysine/chemistry , Melanocytes/radiation effects , Phosphorylation , Protein Processing, Post-Translational , Serine/chemistry , Ultraviolet Rays , Xeroderma Pigmentosum Group A Protein/chemistry
8.
Sci Rep ; 7(1): 11708, 2017 09 15.
Article in English | MEDLINE | ID: mdl-28916831

ABSTRACT

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.


Subject(s)
Cyclic AMP/metabolism , DNA Damage/drug effects , DNA Repair/drug effects , Melanocortins/physiology , Mutagenesis/drug effects , Cell Line , Cells, Cultured , Genomic Instability/drug effects , HEK293 Cells , Humans , Platinum Compounds/toxicity , Signal Transduction/physiology
9.
Sci Rep ; 7(1): 1274, 2017 04 28.
Article in English | MEDLINE | ID: mdl-28455491

ABSTRACT

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.


Subject(s)
Melanocytes/physiology , Melanocytes/radiation effects , Melatonin/metabolism , NF-E2-Related Factor 2/metabolism , Radiation-Protective Agents/metabolism , Ultraviolet Rays , Cells, Cultured , DNA Repair/drug effects , Humans , Reactive Oxygen Species/metabolism , Reactive Oxygen Species/toxicity
10.
Exp Dermatol ; 26(7): 577-584, 2017 07.
Article in English | MEDLINE | ID: mdl-28094871

ABSTRACT

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.


Subject(s)
Cyclic AMP/metabolism , DNA Repair , Melanocytes/cytology , Receptor, Melanocortin, Type 1/metabolism , Skin Pigmentation , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA Damage , Humans , Levodopa/chemistry , Melanins/chemistry , Mutagenesis , Nucleotides/chemistry , Phosphorylation , RNA, Small Interfering/metabolism , Receptor, Melanocortin, Type 1/genetics , Serine/chemistry , Serine/genetics , Signal Transduction , Ultraviolet Rays , Up-Regulation
11.
Photochem Photobiol ; 93(1): 245-258, 2017 01.
Article in English | MEDLINE | ID: mdl-27645605

ABSTRACT

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.


Subject(s)
DNA Repair , Melanocortins/metabolism , Melanocytes/metabolism , Pyrimidine Dimers/metabolism , Signal Transduction , Ultraviolet Rays/adverse effects , Cyclic AMP/metabolism , Humans , Melanoma/epidemiology , Receptor, Melanocortin, Type 1/agonists , Receptor, Melanocortin, Type 1/antagonists & inhibitors , Receptor, Melanocortin, Type 1/metabolism , United States/epidemiology , Xeroderma Pigmentosum/etiology
12.
Nucleic Acids Res ; 44(22): 10711-10726, 2016 12 15.
Article in English | MEDLINE | ID: mdl-27683220

ABSTRACT

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.


Subject(s)
A Kinase Anchor Proteins/physiology , Cell Cycle Proteins/physiology , Cyclic AMP-Dependent Protein Kinases/physiology , Protein Processing, Post-Translational , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Damage , DNA Repair , HEK293 Cells , Humans , Kinetics , Mutagenesis , Phosphorylation , Protein Transport , Xeroderma Pigmentosum Group A Protein/metabolism
13.
J Invest Dermatol ; 135(12): 3086-3095, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26168232

ABSTRACT

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.


Subject(s)
DNA Repair , Melanocytes/metabolism , Receptor, Melanocortin, Type 1/physiology , Agouti Signaling Protein/pharmacology , Ataxia Telangiectasia Mutated Proteins/metabolism , Biomarkers , Cells, Cultured , Cyclic AMP-Dependent Protein Kinases/physiology , DNA/metabolism , Humans , Phosphorylation , Serine/metabolism , Xeroderma Pigmentosum Group A Protein/metabolism , beta-Defensins/pharmacology
14.
Naunyn Schmiedebergs Arch Pharmacol ; 388(2): 199-206, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25017017

ABSTRACT

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.


Subject(s)
Melanoma , NM23 Nucleoside Diphosphate Kinases/metabolism , Skin Neoplasms , Actins/metabolism , Animals , Cell Movement , DNA Repair , Focal Adhesions , Genomic Instability , Humans , Melanoma/genetics , Melanoma/metabolism , Melanoma/pathology , Skin Neoplasms/genetics , Skin Neoplasms/metabolism , Skin Neoplasms/pathology
15.
Mol Cell ; 54(6): 999-1011, 2014 Jun 19.
Article in English | MEDLINE | ID: mdl-24950377

ABSTRACT

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.


Subject(s)
DNA Damage , Receptor, Melanocortin, Type 1/genetics , Xeroderma Pigmentosum Group A Protein/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins/chemistry , Ataxia Telangiectasia Mutated Proteins/genetics , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/genetics , DNA Repair/genetics , DNA-Binding Proteins/genetics , HEK293 Cells , Humans , MCF-7 Cells , Mice , Mice, Inbred C57BL , Mutagenesis/radiation effects , Phosphorylation/radiation effects , Pigmentation/genetics , Protein Processing, Post-Translational/genetics , Protein Processing, Post-Translational/radiation effects , RNA Interference , RNA, Small Interfering , Signal Transduction/genetics , Signal Transduction/radiation effects , Ultraviolet Rays , Xeroderma Pigmentosum Group A Protein/genetics
16.
J Pineal Res ; 57(1): 90-102, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24867336

ABSTRACT

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.


Subject(s)
Keratinocytes/drug effects , Keratinocytes/radiation effects , Melatonin/pharmacology , Ultraviolet Rays , Cell Line , DNA Damage/drug effects , Humans , Kynuramine/pharmacology , Melatonin/analogs & derivatives , Serotonin/analogs & derivatives , Serotonin/pharmacology
18.
Clin Exp Metastasis ; 30(1): 25-36, 2013 Jan.
Article in English | MEDLINE | ID: mdl-22699362

ABSTRACT

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.


Subject(s)
Disease Models, Animal , Genomic Instability , Melanoma/etiology , NM23 Nucleoside Diphosphate Kinases/physiology , Skin Neoplasms/etiology , Ultraviolet Rays/adverse effects , Animals , Cell Movement , DNA Damage/genetics , DNA Repair/genetics , Female , Hepatocyte Growth Factor/genetics , Humans , Hypoxanthine Phosphoribosyltransferase/genetics , Male , Melanoma/pathology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Mutation/genetics , Skin Neoplasms/secondary , Tumor Cells, Cultured , Wound Healing
19.
Methods Mol Biol ; 935: 227-43, 2013.
Article in English | MEDLINE | ID: mdl-23150372

ABSTRACT

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.


Subject(s)
DNA Damage , DNA, Mitochondrial/genetics , Mitochondria/genetics , Mitochondria/metabolism , Real-Time Polymerase Chain Reaction/methods , Retina/cytology , Animals , Cell Culture Techniques/methods , DNA, Mitochondrial/isolation & purification , Humans , Mitochondria/pathology , Oxidative Stress , Oxygen/metabolism , Retina/metabolism , Retina/pathology
20.
Mol Aspects Med ; 33(4): 399-417, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22510306

ABSTRACT

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.


Subject(s)
Macular Degeneration/etiology , Macular Degeneration/metabolism , Oxidative Stress , Animals , Antioxidants/metabolism , Antioxidants/pharmacology , Humans , Macular Degeneration/prevention & control , Oxidative Stress/drug effects , Reactive Nitrogen Species/metabolism , Reactive Oxygen Species/metabolism , Retina/drug effects , Retina/metabolism , Retina/pathology
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