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1.
Cell Death Differ ; 18(8): 1305-15, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21311568

RESUMO

Lamin A is a nuclear lamina constituent expressed in differentiated cells. Mutations in the LMNA gene cause several diseases, including muscular dystrophy and cardiomyopathy. Among the nuclear envelope partners of lamin A are Sad1 and UNC84 domain-containing protein 1 (SUN1) and Sad1 and UNC84 domain-containing protein 2 (SUN2), which mediate nucleo-cytoskeleton interactions critical to the anchorage of nuclei. In this study, we show that differentiating human myoblasts accumulate farnesylated prelamin A, which elicits upregulation and recruitment of SUN1 to the nuclear envelope and favors SUN2 enrichment at the nuclear poles. Indeed, impairment of prelamin A farnesylation alters SUN1 recruitment and SUN2 localization. Moreover, nuclear positioning in myotubes is severely affected in the absence of farnesylated prelamin A. Importantly, reduced prelamin A and SUN1 levels are observed in Emery-Dreifuss muscular dystrophy (EDMD) myoblasts, concomitant with altered myonuclear positioning. These results demonstrate that the interplay between SUN1 and farnesylated prelamin A contributes to nuclear positioning in human myofibers and may be implicated in pathogenetic mechanisms.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Anticolesterolemiantes/farmacologia , Diferenciação Celular , Células Cultivadas , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lamina Tipo A , Lovastatina/farmacologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Mioblastos/citologia , Mioblastos/metabolismo , Prenilação , Células-Tronco/citologia , Células-Tronco/fisiologia
2.
Eur J Histochem ; 55(4): e36, 2011 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-22297442

RESUMO

Farnesylated prelamin A is a processing intermediate produced in the lamin A maturation pathway. Accumulation of a truncated farnesylated prelamin A form, called progerin, is a hallmark of the severe premature ageing syndrome, Hutchinson-Gilford progeria. Progerin elicits toxic effects in cells, leading to chromatin damage and cellular senescence and ultimately causes skin and endothelial defects, bone resorption, lipodystrophy and accelerated ageing. Knowledge of the mechanism underlying prelamin A turnover is critical for the development of clinically effective protein inhibitors that can avoid accumulation to toxic levels without impairing lamin A/C expression, which is essential for normal biological functions. Little is known about specific molecules that may target farnesylated prelamin A to elicit protein degradation. Here, we report the discovery of rapamycin as a novel inhibitor of progerin, which dramatically and selectively decreases protein levels through a mechanism involving autophagic degradation. Rapamycin treatment of progeria cells lowers progerin, as well as wild-type prelamin A levels, and rescues the chromatin phenotype of cultured fibroblasts, including histone methylation status and BAF and LAP2alpha distribution patterns. Importantly, rapamycin treatment does not affect lamin C protein levels, but increases the relative expression of the prelamin A endoprotease ZMPSTE24. Thus, rapamycin, an antibiotic belonging to the class of macrolides, previously found to increase longevity in mouse models, can serve as a therapeutic tool, to eliminate progerin, avoid farnesylated prelamin A accumulation, and restore chromatin dynamics in progeroid laminopathies.


Assuntos
Autofagia/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteínas Nucleares/metabolismo , Progéria/patologia , Precursores de Proteínas/metabolismo , Sirolimo/farmacologia , Antibacterianos/farmacologia , Western Blotting , Células Cultivadas , Criança , Cromatina/metabolismo , Humanos , Lamina Tipo A , Membrana Nuclear/efeitos dos fármacos , Prenilação
3.
Neuromuscul Disord ; 20(8): 512-6, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20580235

RESUMO

Mutations in the lamin A/C gene (LMNA) are known to be involved in several diseases such as Emery-Dreifuss muscular dystrophy, limb-girdle muscular dystrophy type 1B and dilated cardiomyopathies with conduction disease, with considerable phenotype heterogeneity. Here we report on a novel autosomal dominant mutation in LMNA in two direct relatives presenting with different clinical phenotypes, characterized by severe life-threatening limb-girdle muscle involvement and cardiac dysfunction treated with heart transplantation in the proband, and by ventricular tachyarrhythmias with preserved cardiac and skeletal muscle function in her young son. To our knowledge, this is the first report of a duplication in the LMNA gene. The two phenotypes described could reflect different clinical stages of the same disease. We hypothesize that early recognition and initiation of therapeutic manoeuvres in the younger patient may retard the rate of progression of the cardiomyopathy.


Assuntos
Cardiopatias/genética , Cardiopatias/fisiopatologia , Transplante de Coração/fisiologia , Coração/fisiopatologia , Lamina Tipo A/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Adulto , Sequência de Aminoácidos , Cardiomiopatia Dilatada/diagnóstico por imagem , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Eletrocardiografia , Feminino , Duplicação Gênica , Cardiopatias/diagnóstico por imagem , Humanos , Imuno-Histoquímica , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Dados de Sequência Molecular , Debilidade Muscular/genética , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/diagnóstico por imagem , Distrofia Muscular do Cíngulo dos Membros/patologia , Proteínas Nucleares/genética , Linhagem , Fenótipo , Volume Sistólico/fisiologia , Tomografia Computadorizada por Raios X
4.
J Med Genet ; 46(1): 40-8, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18805829

RESUMO

BACKGROUND: Type 2 familial partial lipodystrophy (FPLD2) is characterised by loss of fat in the limbs and buttocks and results from mutations in the LMNA gene. AIM: To evaluate the role of several genes involved in adipogenesis in order to better understand the underlying mechanisms of regional loss of subcutaneous adipose tissue (scAT) in patients with FPLD2. METHODS: In total, 7 patients with FPLD2 and 10 healthy control participants were studied. A minimal model was used to calculate the insulin sensitivity (IS). scAT was obtained from abdomen and thigh by biopsy. Relative gene expression was quantified by real-time reverse transcription PCR in a thermal cycler. Prelamin A western blot analysis was carried out on scAT and prelamin A nuclear localisation was determined using immunofluorescence. Adipocyte nuclei were examined by electron microscopy. RESULTS: Patients with FPLD2 were found to have significantly lower IS. The expression of LMNA was similar in both groups. The expression of PPARG2, RB1, CCND3 and LPL in thigh but not in abdomen scAT was significantly reduced (67%, 25%, 38% and 66% respectively) in patients with FPLD2. Significantly higher levels of prelamin A were found in peripheral scAT of patients with FPLD2. Defects in the peripheral heterochromatin and a nuclear fibrous dense lamina were present in the adipocytes of patients with FPLD2. CONCLUSIONS: In FPLD2 participants, prelamin A accumulation in peripheral scAT is associated with a reduced expression of several genes involved in adipogenesis, which could perturb the balance between proliferation and differentiation in adipocytes, leading to less efficient tissue regeneration.


Assuntos
Lipodistrofia Parcial Familiar/genética , Proteínas Nucleares/genética , Precursores de Proteínas/genética , Gordura Subcutânea/patologia , Adipogenia/genética , Tecido Adiposo/patologia , Adulto , Feminino , Imunofluorescência , Genes Reguladores , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia Parcial Familiar/patologia , Masculino , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Precursores de Proteínas/metabolismo , Gordura Subcutânea/ultraestrutura
5.
Eur J Histochem ; 50(1): 1-8, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16584978

RESUMO

The involvement of the nuclear envelope in the modulation of chromatin organization is strongly suggested by the increasing number of human diseases due to mutations of nuclear envelope proteins. A common feature of these diseases, named laminopathies, is the occurrence of major chromatin defects. We previously reported that cells from laminopathic patients show an altered nuclear profile, and loss or detachment of heterochromatin from the nuclear envelope. Recent evidence indicates that processing of the lamin A precursor is altered in laminopathies featuring pre-mature aging and/or lipodystrophy phenotype. In these cases, pre-lamin A is accumulated in the nucleus and heterochromatin is severely disorganized. Here we report evidence indicating that pre-lamin A is mis-localized in the nuclei of Emery-Dreifuss muscular dystrophy fibroblasts, either bearing lamin A/C or emerin mutations. Abnormal pre-lamin A-containing structures are formed following treatment with a farnesyl-transferase inhibitor, a drug that causes accumulation of pre-lamin A. Pre-lamin A-labeled structures co-localize with heterochromatin clumps. These data indicate that in almost all laminopathies the expression of the mutant lamin A precursor disrupts the organization of heterochromatin domains. Our results further show that the absence of emerin expression alters the distribution of pre-lamin A and of heterochromatin areas, suggesting a major involvement of emerin in pre-lamin A-mediated mechanisms of chromatin remodeling.


Assuntos
Montagem e Desmontagem da Cromatina , Heterocromatina , Lamina Tipo A , Membrana Nuclear/metabolismo , Senilidade Prematura/genética , Animais , Montagem e Desmontagem da Cromatina/genética , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lipodistrofia/genética , Lipodistrofia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mutação , Proteínas Nucleares , Timopoietinas/genética , Timopoietinas/metabolismo
6.
Eur J Histochem ; 49(4): 355-62, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-16377577

RESUMO

The fate of emerin during skeletal muscle regeneration was investigated in an animal model by means of crush injury. Immunofluorescence, immunoblotting and mRNA analysis demonstrated that emerin level is increased in regenerating rat muscle fibers with respect to normal mature myofibers. This finding suggests an involvement of emerin during the muscle fiber regeneration process, in analogy with its reported involvement in muscle cell differentiation in vitro. The impairment of skeletal muscle physiological regeneration or reorganization could be a possible pathogenetic mechanism for Emery Dreifuss muscular dystrophy.


Assuntos
Proteínas de Membrana/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/fisiologia , Regeneração/fisiologia , Timopoietinas/metabolismo , Animais , Diferenciação Celular/fisiologia , Núcleo Celular/metabolismo , Células Cultivadas , Imunofluorescência , Immunoblotting , Imuno-Histoquímica , Masculino , Modelos Animais , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas Nucleares , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Regulação para Cima
7.
Cell Mol Life Sci ; 62(22): 2669-78, 2005 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16261260

RESUMO

Hutchinson-Gilford progeria (HGPS) is a premature aging syndrome associated with LMNA mutations. Progeria cells bearing the G608G LMNA mutation are characterized by accumulation of a mutated lamin A precursor (progerin), nuclear dysmorphism and chromatin disorganization. In cultured HGPS fibroblasts, we found worsening of the cellular phenotype with patient age, mainly consisting of increased nuclear-shape abnormalities, progerin accumulation and heterochromatin loss. Moreover, transcript distribution was altered in HGPS nuclei, as determined by different techniques. In the attempt to improve the cellular phenotype, we applied treatment with drugs either affecting protein farnesylation or chromatin arrangement. Our results show that the combined treatment with mevinolin and the histone deacetylase inhibitor trichostatin A dramatically lowers progerin levels, leading to rescue of heterochromatin organization and reorganization of transcripts in HGPS fibroblasts. These results suggest that morpho-functional defects of HGPS nuclei are directly related to progerin accumulation and can be rectified by drug treatment.


Assuntos
Heterocromatina/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Progéria/tratamento farmacológico , Progéria/genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Células Cultivadas , Criança , Metilação de DNA , Heterocromatina/ultraestrutura , Histonas/metabolismo , Humanos , Lamina Tipo A/deficiência , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Lovastatina/farmacologia , Progéria/metabolismo , Progéria/patologia , Precursores de Proteínas/metabolismo , Ribonucleoproteínas/metabolismo
8.
J Med Genet ; 42(3): 214-20, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15744034

RESUMO

BACKGROUND: Skeletal muscle disorders associated with mutations of lamin A/C gene include autosomal Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B. The pathogenic mechanism underlying these diseases is unknown. Recent data suggest an impairment of signalling mechanisms as a possible cause of muscle malfunction. A molecular complex in muscle cells formed by lamin A/C, emerin, and nuclear actin has been identified. The stability of this protein complex appears to be related to phosphorylation mechanisms. OBJECTIVE: To analyse lamin A/C phosphorylation in control and laminopathic muscle cells. METHODS: Lamin A/C N-terminal phosphorylation was determined in cultured mouse myoblasts using a specific antibody. Insulin treatment of serum starved myoblast cultures was carried out to evaluate involvement of insulin signalling in the phosphorylation pathway. Screening of four Emery-Dreifuss and one limb girdle muscular dystrophy 1B cases was undertaken to investigate lamin A/C phosphorylation in both cultured myoblasts and mature muscle fibres. RESULTS: Phosphorylation of lamin A was observed during myoblast differentiation or proliferation, along with reduced lamin A/C phosphorylation in quiescent myoblasts. Lamin A N-terminus phosphorylation was induced by an insulin stimulus, which conversely did not affect lamin C phosphorylation. Lamin A/C was also hyperphosphorylated in mature muscle, mostly in regenerating fibres. Lamin A/C phosphorylation was strikingly reduced in laminopathic myoblasts and muscle fibres, while it was preserved in interstitial fibroblasts. CONCLUSIONS: Altered lamin A/C interplay with a muscle specific phosphorylation partner might be involved in the pathogenic mechanism of Emery-Dreifuss muscular dystrophy and limb girdle muscular dystrophy 1B.


Assuntos
Lamina Tipo A/metabolismo , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mioblastos/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Diferenciação Celular , Linhagem Celular , Humanos , Insulina/metabolismo , Lamina Tipo A/genética , Camundongos , Fibras Musculares Esqueléticas/metabolismo , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/metabolismo , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular de Emery-Dreifuss/genética , Fosforilação , Transdução de Sinais
9.
Eur J Histochem ; 47(1): 3-16, 2003.
Artigo em Inglês | MEDLINE | ID: mdl-12685553

RESUMO

The present review summarizes recent cytochemical findings on the functional organization of the nuclear domains, with a particular emphasis on the relation between nuclear envelope-associated proteins and chromatin. Mutations in two nuclear envelope-associated proteins, emerin and lamin A/C cause the Emery-Dreifuss muscular dystrophy; the cellular pathology associated with the disease and the functional role of emerin and lamin A/C in muscle cells are not well established. On the other hand, a large body of evidence indicates that nuclear envelope-associated proteins are involved in tissue-specific gene regulation. Moreover, chromatin remodeling complexes trigger gene expression by utilizing the nuclear matrix-associated actin, which is known to interact with both emerin and lamin A/C. It is thus conceivable that altered expression of these nuclear envelope-associated proteins can account for an impairment of gene expression mainly during cell differentiation as suggested by recent experimental findings on the involvement of emerin in myogenesis. The possibility that Emery-Deifuss muscular dystrophy pathogenesis could involve alteration of the signaling pathway is considered.


Assuntos
Imuno-Histoquímica , Distrofia Muscular de Emery-Dreifuss/metabolismo , Distrofia Muscular de Emery-Dreifuss/fisiopatologia , Membrana Nuclear/metabolismo , Expressão Gênica , Humanos , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Membrana Nuclear/genética , Proteínas Nucleares , Transdução de Sinais , Timopoietinas/genética , Timopoietinas/metabolismo
10.
FEBS Lett ; 509(3): 423-9, 2001 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-11749967

RESUMO

Emerin is a nuclear membrane-anchored protein which is absent or mutated in patients affected by Emery-Dreifuss muscular dystrophy. In this study, we induced apoptosis in cultured mouse myoblasts to evaluate emerin fate during the nuclear destabilization involved in programmed cell death. Emerin proteolysis was observed in myocytes during the apoptotic process. Myoblast apoptosis and emerin degradation were associated with chromatin compaction and detachment from the nuclear lamina, as detected by electron microscopy. In vivo specific inhibition of caspase 3 or caspase 6 activity completely abolished emerin proteolysis. These results show that the process of programmed cell death in muscle cells leads to emerin proteolysis, which appears to be related to caspase 6 activation and to cleavage of other nuclear envelope proteins, that share sequence homologies or functional features with emerin.


Assuntos
Caspases/metabolismo , Proteínas de Membrana/metabolismo , Músculos/efeitos dos fármacos , Músculos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Estaurosporina/farmacologia , Timopoietinas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Caspase 3 , Inibidores de Caspase , Linhagem Celular , Meios de Cultura Livres de Soro , Cinética , Camundongos , Microscopia Eletrônica , Microscopia de Fluorescência , Músculos/enzimologia , Músculos/ultraestrutura , Proteínas Nucleares , Fatores de Tempo
11.
Matrix Biol ; 20(7): 475-86, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11691587

RESUMO

Fibronectin is one of the main components of the extracellular matrix and associates with a variety of other matrix molecules including collagens. We demonstrate that the absence of secreted type VI collagen in cultured primary fibroblasts affects the arrangement of fibronectin in the extracellular matrix. We observed a fine network of collagen VI filaments and fibronectin fibrils in the extracellular matrix of normal murine and human fibroblasts. The two microfibrillar systems did not colocalize, but were interconnected at some discrete sites which could be revealed by immunoelectron microscopy. Direct interaction between collagen VI and fibronectin was also demonstrated by far western assay. When primary fibroblasts from Col6a1 null mutant mice were cultured, collagen VI was not detected in the extracellular matrix and a different pattern of fibronectin organization was observed, with fibrils running parallel to the long axis of the cells. Similarly, an abnormal fibronectin deposition was observed in fibroblasts from a patient affected by Bethlem myopathy, where collagen VI secretion was drastically reduced. The same pattern was also observed in normal fibroblasts after in vivo perturbation of collagen VI-fibronectin interaction with the 3C4 anti-collagen VI monoclonal antibody. Competition experiments with soluble peptides indicated that the organization of fibronectin in the extracellular matrix was impaired by added soluble collagen VI, but not by its triple helical (pepsin-resistant) fragments. These results indicate that collagen VI mediates the three-dimensional organization of fibronectin in the extracellular matrix of cultured fibroblasts.


Assuntos
Colágeno Tipo VI/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Colágeno Tipo VI/genética , Fibroblastos/citologia , Humanos , Camundongos , Camundongos Knockout , Camundongos Nus
12.
Muscle Nerve ; 24(6): 826-9, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11360268

RESUMO

Electron microscopy study of muscle biopsies from patients with autosomal-dominant Emery-Dreifuss muscular dystrophy revealed nuclear alterations in about 10% of the preserved muscle fibers. The major findings consisted of peripheral heterochromatin loss or detachment from the nuclear envelope, and of interchromatin texture alterations. These abnormalities are similar to those reported in an animal model of the disease and to those found in the X-linked form of Emery-Dreifuss muscular dystrophy. These results suggest that an abnormal ultrastructural arrangement of the nuclear periphery is a common feature in the known forms of Emery-Dreifuss muscular dystrophy, and that several proteins of the nuclear scaffold are necessary in muscle cells to maintain the nuclear structural/functional integrity and a normal muscle cell metabolism.


Assuntos
Núcleo Celular/patologia , Músculo Esquelético/patologia , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/patologia , Adulto , Substituição de Aminoácidos , Biópsia , Núcleo Celular/ultraestrutura , Criança , Feminino , Heterocromatina/patologia , Heterocromatina/ultraestrutura , Humanos , Laminas , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/patologia , Fibras Musculares Esqueléticas/ultraestrutura , Músculo Esquelético/ultraestrutura , Proteínas Nucleares/genética , Cromossomo X
13.
Biochem Biophys Res Commun ; 277(3): 639-42, 2000 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-11062006

RESUMO

We recently described a novel congenital muscular dystrophy (CMD) syndrome characterized by mental retardation, microcephaly, and partial merosin deficiency on muscle biopsy. Linkage analysis excluded involvement of the known CMD loci. We now report on a study performed on the differentiation of cultured myoblasts from one patient affected by this condition to evaluate the potential to form myotubes and merosin processing in these cells. The differentiation rate was comparable to controls and myotubes were stable in culture. Biochemical analysis showed the expected 80-kDa merosin subunit in myoblasts. However, a shifted 60-kDa protein was detected in myotubes. Matrix-metalloproteinases (MMPs) zymography showed increased gelatinolytic activity, and immunoblotting identified an increased amount of membrane-type 1 matrix-metalloproteinase in pathological myotube preparations. Our results show that these CMD-derived myotubes contain a low molecular weight merosin. They further suggest that an altered regulation of MMPs can be involved in basal lamina damage.


Assuntos
Laminina/metabolismo , Distrofias Musculares/metabolismo , Diferenciação Celular , Pré-Escolar , Feminino , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinases da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/metabolismo , Distrofias Musculares/enzimologia , Distrofias Musculares/patologia
14.
Histochem J ; 32(2): 115-22, 2000 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10816077

RESUMO

2',3'-Dideoxycytidine is a powerful in vitro inhibitor of human immunodeficiency virus and is currently used in the treatment of acquired immunodeficiency syndrome. A long-term exposure of U937 monoblastoid cells to dideoxycytidine induces the selection of drug-resistant cells (U937-R). In previous studies, we investigated some important biochemical properties and functional activities, such as basal respiration, protein kinase C activity, superoxide anion release, and the level of reduced glutathione, which were found to be higher in the drug-resistant cell line, compared to the parental one. In the present study, we evaluated the response of the two cell lines to the induction of apoptosis by treatment with staurosporine and okadaic acid, which interfere with the protein kinase and phosphatase pathways, respectively. Moreover, knowing that GSH plays a crucial role in the regulation of nitric oxide-dependent apoptosis, U937-R and parental lines have been treated with SIN-1, which is known to generate significant amounts of O2 and nitric oxide. Resistant and parental cells have been analysed by light and electron microscopy and agarose gel electrophoresis of isolated DNA has been performed. The obtained results demonstrate a different susceptibility of U937-R cell line to apoptosis induced with the three triggers. U937-R cells show more advanced apoptotic features if compared with parental cells, after staurosporine treatment. Differently, the okadaic acid does not induce a different behaviour in the two models. On the contrary, the agent SIN-1 determines an increased number of apoptotic cells in the U937 line. The results suggest that a higher level of protein kinase C and glutathione could prevent programmed cell death in U937-R.


Assuntos
Fármacos Anti-HIV/farmacologia , Apoptose/efeitos dos fármacos , Zalcitabina/farmacologia , DNA/análise , Resistência a Medicamentos , Eletroforese em Gel de Ágar , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Humanos , Molsidomina/análogos & derivados , Molsidomina/farmacologia , Doadores de Óxido Nítrico/farmacologia , Ácido Okadáico/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Inibidores de Proteínas Quinases , Estaurosporina/farmacologia , Células U937/efeitos dos fármacos , Células U937/metabolismo , Células U937/ultraestrutura
15.
Differentiation ; 66(4-5): 208-17, 2000 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11269947

RESUMO

Emerin is an ubiquitous protein localized at the nuclear membrane of most cell types including muscle cells. The protein is absent in most patients affected by the X-linked form of Emery-Dreifuss muscular dystrophy, a disease characterized by slowly progressive muscle wasting and weakness, early contractures of the elbows, Achilles tendons, and post-cervical muscles, and cardiomyopathy. Besides the nuclear localization, emerin cytoplasmic distribution has been suggested in several cell types. We studied the expression and the subcellular distribution of emerin in mouse cultured C2C12 myoblasts and in primary cultures of human myoblasts induced to differentiate or spontaneously differentiating in the culture medium. In differentiating myoblasts transiently transfected with a cDNA encoding the complete emerin sequence, the protein localized at the nuclear rim of all transfected cells and also in the cytoplasm of some myoblasts and myotubes. Cytoplasmic emerin was also observed in detergent-treated myotubes, as determined by electron microscopy observation. Both immunofluorescence and biochemical analysis showed, that upon differentiation of C2C12 cells, emerin expression was decreased in the resting myoblasts but the protein was highly represented in the developing myotubes at the early stage of cell fusion. Labeling with specific markers of myogenesis such as troponin-T and myogenin permitted the correlation of increased emerin expression with the onset of muscle differentiation. These data suggest a role for emerin during proliferation of activated satellite cells and at the early stages of differentiation.


Assuntos
Proteínas de Membrana/biossíntese , Músculo Esquelético/citologia , Timopoietinas/biossíntese , Animais , Western Blotting , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Linhagem Celular , Citoplasma/metabolismo , Citoesqueleto/metabolismo , DNA Complementar/genética , Epitopos/genética , Citometria de Fluxo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Microscopia Eletrônica , Microtúbulos/metabolismo , Músculo Esquelético/metabolismo , Proteínas Nucleares , Frações Subcelulares/metabolismo , Timopoietinas/genética , Timopoietinas/imunologia , Transfecção
16.
Biochem Pharmacol ; 58(11): 1743-50, 1999 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-10571248

RESUMO

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.


Assuntos
Apoptose , Benzamidas/farmacologia , Inibidores Enzimáticos/farmacologia , Peróxido de Hidrogênio/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Nucleotídeos de Adenina/metabolismo , DNA/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Interações Medicamentosas , Humanos , Necrose , Oxidantes/farmacologia , Células U937
17.
Scanning ; 21(1): 29-35, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10070781

RESUMO

Molt-4 human leukemia cells were triggered to apoptosis by various agents with different mechanisms of action. Staurosporine, a protein kinase C (PKC) inhibitor; camptothecin, a topoisomerase I blocking drug; and tiazofurin, an inhibitor of inosine 5'-phosphate dehydrogenase (IMPDH), were used. Ultrastructural analysis showed morphologic changes characteristic of apoptosis that were very similar for all three agents. Nevertheless, DNA oligonucleosomic fragmentation was not detectable by agarose gel electrophoresis. However, a genomic DNA cleavage appeared after pulse-field gel electrophoresis (PFGE) in cells treated with these agents for 24 h. Furthermore, in situ nick translation (NT) showed a finely spotted nuclear labelling in staurosporine-treated cells and a compact fluorescence after camptothecin incubation. In tiazofurin-treated cells an intermediate pattern was found. Therefore, apoptotic agents with different mechanisms of action induced the formation of large genomic DNA fragments and very similar ultrastructural changes. Therefore, both phenomena and the closely related apoptosis progression depend on target cell machinery and not on the triggering agent.


Assuntos
Apoptose , Núcleo Celular/efeitos dos fármacos , Fragmentação do DNA , Inibidores Enzimáticos/farmacologia , Camptotecina/farmacologia , Núcleo Celular/ultraestrutura , Eletroforese em Gel de Ágar , Eletroforese em Gel de Campo Pulsado , Técnicas Genéticas , Humanos , IMP Desidrogenase/antagonistas & inibidores , Leucemia Linfoide , Proteína Quinase C/antagonistas & inibidores , Ribavirina/análogos & derivados , Ribavirina/farmacologia , Estaurosporina/farmacologia , Inibidores da Topoisomerase I , Células Tumorais Cultivadas
18.
Anat Rec ; 254(1): 1-6, 1999 01.
Artigo em Inglês | MEDLINE | ID: mdl-9892411

RESUMO

Apoptosis plays a fundamental role in shaping normal hematopoiesis. We have investigated the relationship existing between susceptibility to apoptosis and lineage commitment in hemopoietic cells. The presence and degree of apoptosis were investigated in myeloid (HL-60 and K562), T (Jurkat and MOLT-4), and B (CESS and Raji) lymphoid cell lines by using a variety of techniques-transmission electron and light microscopy, flow cytometry and DNA gel electrophoresis. The major achievement of this study is that hematopoietic cells respond to different chemical (staurosporin, tiazofurin, camptothecin) and physical (hyperthermia or hypothermia) stimuli by apoptosis in a lineage-related way. Moreover, with respect to the methods used to detect apoptosis, a strong correlation was observed between the presence of the hypodiploid peak determined by flow cytometry and the DNA laddering evaluated by gel electrophoresis, but both techniques failed to demonstrate the presence of apoptosis in some cases. We conclude that cells of different hematopoietic lineages mostly show a lineage-related behaviour in their apoptotic response to different stimuli, suggesting that the lineage commitment and the stage of differentiation can confer different sensitivities to specific apoptotic stimuli. Moreover, morphological techniques still represent the most reliable approach to detect apoptosis in hemopoietic cells.


Assuntos
Apoptose/fisiologia , Hematopoese/fisiologia , Sistema Hematopoético/citologia , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Linhagem Celular , Temperatura Baixa , Fragmentação do DNA/efeitos dos fármacos , Hematopoese/efeitos dos fármacos , Sistema Hematopoético/efeitos dos fármacos , Temperatura Alta , Humanos , Microscopia Eletrônica , Ribavirina/análogos & derivados , Ribavirina/farmacologia , Estaurosporina/farmacologia
19.
Histochem Cell Biol ; 110(5): 467-76, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9826126

RESUMO

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.


Assuntos
Apoptose , Membrana Celular/ultraestrutura , Fosfolipídeos/química , Membrana Celular/química , Membrana Celular/patologia , Fluorescência , Células HL-60 , Humanos
20.
J Submicrosc Cytol Pathol ; 30(2): 285-93, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9648292

RESUMO

A study was performed to evaluate the effect of prolonged environmental hypobaric hypoxia on the ultrastructure of rat cornea. 60-day-old female Wistar rats were exposed to a simulated altitude of 5,500 m (350 mmHg) and pO2 of 76 mmHg for 30 days. Control rats were exposed to atmospheric sea level pressure (760 mmHg) and pO2 of 150 mmHg, for the same period. Ultrastructural analysis of the corneal epithelium did not reveal any relevant difference between control and treated rats. In contrast, the corneal stroma of rats subjected to hypoxia showed vascularization with advanced vessel differentiation and signs of active proliferation. The endothelium of hypoxic cornea showed swollen mitochondria and large empty cytoplasmic areas. The endothelial intercellular junctions could hardly be identified in the hypoxic condition. Nevertheless, the most evident change in hypoxic cornea was in Descemet's membrane, which was considerably thickened, to approximately twice that of the control specimen. These results suggest that environmental hypobaric hypoxia induces marked alterations in the corneal stroma and endothelium morphology, probably related to reduced oxygen tension in the aqueous humor, consequent to hypoxemia.


Assuntos
Córnea/ultraestrutura , Oxigênio , Animais , Pressão Atmosférica , Hipóxia Celular , Substância Própria/ultraestrutura , Lâmina Limitante Posterior/ultraestrutura , Endotélio Corneano/ultraestrutura , Epitélio Corneano/ultraestrutura , Feminino , Linfócitos/ultraestrutura , Neutrófilos/ultraestrutura , Ratos , Ratos Wistar
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