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1.
J Membr Biol ; 249(4): 569-76, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27312328

RESUMO

Electroporation-based treatments and other therapies that permeabilize the plasma membrane have been shown to be more devastating to malignant cells than to normal cells. In this study, we asked if a difference in repair capacity could explain this observed difference in sensitivity. Membrane repair was investigated by disrupting the plasma membrane using laser followed by monitoring fluorescent dye entry over time in seven cancer cell lines, an immortalized cell line, and a normal primary cell line. The kinetics of repair in living cells can be directly recorded using this technique, providing a sensitive index of repair capacity. The normal primary cell line of all tested cell lines exhibited the slowest rate of dye entry after laser disruption and lowest level of dye uptake. Significantly, more rapid dye uptake and a higher total level of dye uptake occurred in six of the seven tested cancer cell lines (p < 0.05) as well as the immortalized cell line (p < 0.001). This difference in sensitivity was also observed when a viability assay was performed one day after plasma membrane permeabilization by electroporation. Viability in the primary normal cell line (98 % viable cells) was higher than in the three tested cancer cell lines (81-88 % viable cells). These data suggest more effective membrane repair in normal, primary cells and supplement previous explanations why electroporation-based therapies and other therapies permeabilizing the plasma membrane are more effective on malignant cells compared to normal cells in cancer treatment.


Assuntos
Membrana Celular/fisiologia , Regeneração , Linhagem Celular , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular , Eletroquimioterapia , Eletroporação , Humanos , Melanoma/patologia , Melanoma/terapia
2.
Free Radic Biol Med ; 84: 246-253, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25843658

RESUMO

Vitamin E (VE) deficiency results in pronounced muscle weakness and atrophy but the cell biological mechanism of the pathology is unknown. We previously showed that VE supplementation promotes membrane repair in cultured cells and that oxidants potently inhibit repair. Here we provide three independent lines of evidence that VE is required for skeletal muscle myocyte plasma membrane repair in vivo. We also show that when another lipid-directed antioxidant, glutathione peroxidase 4 (Gpx4), is genetically deleted in mouse embryonic fibroblasts, repair fails catastrophically, unless cells are supplemented with VE. We conclude that lipid-directed antioxidant activity provided by VE, and possibly also Gpx4, is an essential component of the membrane repair mechanism in skeletal muscle. This work explains why VE is essential to muscle health and identifies VE as a requisite component of the plasma membrane repair mechanism in vivo.


Assuntos
Antioxidantes/farmacologia , Membrana Celular/metabolismo , Músculo Esquelético/fisiologia , Vitamina E/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Glutationa Peroxidase/metabolismo , Masculino , Camundongos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Ratos Sprague-Dawley
3.
Nat Commun ; 2: 597, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22186893

RESUMO

Severe vitamin E deficiency results in lethal myopathy in animal models. Membrane repair is an important myocyte response to plasma membrane disruption injury as when repair fails, myocytes die and muscular dystrophy ensues. Here we show that supplementation of cultured cells with α-tocopherol, the most common form of vitamin E, promotes plasma membrane repair. Conversely, in the absence of α-tocopherol supplementation, exposure of cultured cells to an oxidant challenge strikingly inhibits repair. Comparative measurements reveal that, to promote repair, an anti-oxidant must associate with membranes, as α-tocopherol does, or be capable of α-tocopherol regeneration. Finally, we show that myocytes in intact muscle cannot repair membranes when exposed to an oxidant challenge, but show enhanced repair when supplemented with vitamin E. Our work suggests a novel biological function for vitamin E in promoting myocyte plasma membrane repair. We propose that this function is essential for maintenance of skeletal muscle homeostasis.


Assuntos
Membrana Celular/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Mioblastos/metabolismo , Deficiência de Vitamina E/sangue , Animais , Membrana Celular/fisiologia , Relação Dose-Resposta a Droga , Glucose/efeitos adversos , Células HeLa , Homeostase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Microscopia de Fluorescência , Fibras Musculares Esqueléticas/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Estresse Oxidativo , Cicatrização/efeitos dos fármacos , alfa-Tocoferol/sangue , alfa-Tocoferol/farmacologia
4.
Diabetes ; 60(11): 3034-43, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21940783

RESUMO

OBJECTIVE: Skeletal muscle myopathy is a common diabetes complication. One possible cause of myopathy is myocyte failure to repair contraction-generated plasma membrane injuries. Here, we test the hypothesis that diabetes induces a repair defect in skeletal muscle myocytes. RESEARCH DESIGN AND METHODS: Myocytes in intact muscle from type 1 (INS2(Akita+/-)) and type 2 (db/db) diabetic mice were injured with a laser and dye uptake imaged confocally to test repair efficiency. Membrane repair defects were also assessed in diabetic mice after downhill running, which induces myocyte plasma membrane disruption injuries in vivo. A cell culture model was used to investigate the role of advanced glycation end products (AGEs) and the receptor for AGE (RAGE) in development of this repair defect. RESULTS: Diabetic myocytes displayed significantly more dye influx after laser injury than controls, indicating a repair deficiency. Downhill running also resulted in a higher level of repair failure in diabetic mice. This repair defect was mimicked in cultured cells by prolonged exposure to high glucose. Inhibition of the formation of AGE eliminated this glucose-induced repair defect. However, a repair defect could be induced, in the absence of high glucose, by enhancing AGE binding to RAGE, or simply by increasing cell exposure to AGE. CONCLUSIONS: Because one consequence of repair failure is rapid cell death (via necrosis), our demonstration that repair fails in diabetes suggests a new mechanism by which myopathy develops in diabetes.


Assuntos
Membrana Celular/metabolismo , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/complicações , Fibras Musculares Esqueléticas/metabolismo , Doenças Musculares/metabolismo , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/efeitos da radiação , Membrana Celular/ultraestrutura , Células Cultivadas , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/toxicidade , Produtos Finais de Glicação Avançada/efeitos adversos , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Produtos Finais de Glicação Avançada/metabolismo , Hiperglicemia/metabolismo , Lasers/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Atividade Motora , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos da radiação , Fibras Musculares Esqueléticas/ultraestrutura , Doenças Musculares/patologia , Mioblastos Esqueléticos/metabolismo , Necrose , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/metabolismo
5.
J Biol Chem ; 281(46): 35202-7, 2006 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-16984915

RESUMO

Ca2+ entering a cell through a torn or disrupted plasma membrane rapidly triggers a combination of homotypic and exocytotic membrane fusion events. These events serve to erect a reparative membrane patch and then anneal it to the defect site. Annexin A1 is a cytosolic protein that, when activated by micromolar Ca2+, binds to membrane phospholipids, promoting membrane aggregation and fusion. We demonstrate here that an annexin A1 function-blocking antibody, a small peptide competitor, and a dominant-negative annexin A1 mutant protein incapable of Ca2+ binding all inhibit resealing. Moreover, we show that, coincident with a resealing event, annexin A1 becomes concentrated at disruption sites. We propose that Ca2+ entering through a disruption locally induces annexin A1 binding to membranes, initiating emergency fusion events whenever and wherever required.


Assuntos
Anexina A1/metabolismo , Membrana Celular/metabolismo , Cálcio/metabolismo , Regulação da Expressão Gênica , Células HeLa , Humanos , Membranas Intracelulares , Micromanipulação , Mutação , Transporte Proteico
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