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1.
J Pharmacol Exp Ther ; 301(2): 753-64, 2002 May.
Artigo em Inglês | MEDLINE | ID: mdl-11961082

RESUMO

(-)-Deprenyl and structurally related propargylamines increase neuronal survival independently of monoamine oxidase B (MAO-B) inhibition, in part by decreasing apoptosis. We found that deprenyl and two other propargylamines, one of which does not inhibit monoamine oxidase B, increased survival in trophically withdrawn 6-day nerve growth factor (NGF)- and 9-day NGF-differentiated PC-12 cells but not in NGF naive or 3-day NGF-differentiated PC-12 cells. Four days of prior NGF exposure were required for the propargylamine-mediated antiapoptosis. Studies using actinomycin D, cycloheximide, and camptothecin revealed that the maintenance of both transcription and translation, particularly between 2 and 6 h after trophic withdrawal, was required for propargylamine-mediated antiapoptosis. Metabolic labeling of newly synthesized proteins for two-dimensional protein gel autoradiography and scintillation counting showed that the propargylamines either increased or reduced the levels of new synthesis or induced de novo synthesis of a number of different proteins, most notably proteins in the mitochondrial and nuclear subfractions. Western blotting for whole cell or subcellular fraction lysates showed that the timing of new protein synthesis changes or subcellular redistribution of apoptosis-related proteins induced by the propargylamines were appropriate to antiapoptosis. The apoptosis-related proteins included superoxide dismutases (SOD1 and SOD2), glutathione peroxidase, c-JUN, and glyceraldehyde-3-phosphate dehydrogenase. Most notable were the prevention of apoptotic decreases in BCL-2 levels and increases in mitochondrial BAX levels. In general, (-)-deprenyl-related propargylamines appear to reduce apoptosis by altering the levels or subcellular localization of proteins that affect mitochondrial membrane permeability, scavenge oxidative radicals, or participate in specific apoptosis signaling pathways.


Assuntos
Apoptose/fisiologia , Fator de Crescimento Neural/metabolismo , Pargilina/análogos & derivados , Pargilina/farmacologia , Propilaminas/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura , Meios de Cultura Livres de Soro , Células PC12 , Inibidores da Síntese de Proteínas/farmacologia , Ratos
2.
J Biol Chem ; 276(23): 19945-53, 2001 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-11262418

RESUMO

A prominent pathway of transforming growth factor (TGF)-beta signaling involves receptor-dependent phosphorylation of Smad2 and Smad3, which then translocate to the nucleus to activate transcription of target genes. To investigate the relative importance of these two Smad proteins in TGF-beta1 signal transduction, we have utilized a loss of function approach, based on analysis of the effects of TGF-beta1 on fibroblasts derived from mouse embryos deficient in Smad2 (S2KO) or Smad3 (S3KO). TGF-beta1 caused 50% inhibition of cellular proliferation in wild-type fibroblasts as assessed by [(3)H]thymidine incorporation, whereas the growth of S2KO or S3KO cells was only weakly inhibited by TGF-beta1. Lack of Smad2 or Smad3 expression did not affect TGF-beta1-induced fibronectin synthesis but resulted in markedly suppressed induction of plasminogen activator inhibitor-1 by TGF-beta1. Moreover, TGF-beta1-mediated induction of matrix metalloproteinase-2 was selectively dependent on Smad2, whereas induction of c-fos, Smad7, and TGF-beta1 autoinduction relied on expression of Smad3. Investigation of transcriptional activation of TGF-beta-sensitive reporter genes in the different fibroblasts showed that activation of the (Smad binding element)(4)-Lux reporter by TGF-beta1 was dependent on expression of Smad3, but not Smad2, whereas activation of the activin response element-Lux reporter was strongly suppressed in S2KO fibroblasts but, on the contrary, enhanced in S3KO cells. Our findings indicate specific roles for Smad2 and Smad3 in TGF-beta1 signaling.


Assuntos
Proteínas de Ciclo Celular , Proteínas de Ligação a DNA/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Proteínas Supressoras de Tumor , Animais , Divisão Celular , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Proteínas de Ligação a DNA/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Precoces , Genes Reporter , Genes fos , Camundongos , Camundongos Knockout , Proteína Smad2 , Proteína Smad3 , Transativadores/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador beta/biossíntese
3.
J Neurosci ; 18(3): 932-47, 1998 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9437015

RESUMO

Studies in non-neural cells have suggested that a fall in mitochondrial membrane potential (DeltaPsiM) is one of the earliest events in apoptosis. It is not known whether neural apoptosis caused by nerve growth factor (NGF) and serum withdrawal involves a decrease in DeltaPsiM. We used epifluorescence and laser confocal microscopy with the mitochondrial potentiometric dyes chloromethyl-tetramethylrosamine methyl ester and 5,5',6, 6'-tetrachloro-1,1',3,3'-tetraethybenzimidazol carbocyanine iodide to estimate DeltaPsiM. PC12 cells were differentiated in media containing serum and NGF for 6 d before withdrawal of trophic support. After washing, the cells were incubated with media containing serum and NGF (M/S+N), media without serum and NGF, or media with the "trophic-like" monoamine oxidase B inhibitor, (-)-deprenyl. Mitochondria in cells without trophic support underwent a progressive shift to lower DeltaPsiM values that was significant by 3 hr after washing. The percentages of cells with nuclear chromatin condensation or nuclear DNA fragmentation were not significantly increased above those for cells in M/S+N until 6 hr after washing. Replacement of cells into M/S+N or treatment with (-)-deprenyl markedly reduced the proportion of mitochondria with decreased DeltaPsiM. Measurements of cytoplasmic peroxyl radical levels with 2',7'-dihydrodichlorofluorescein fluorescence and intramitochondrial Ca2+ with dihydro-rhodamine-2-acetylmethyl ester indicated that cytoplasmic peroxyl radical levels were not increased until after 6 hr, whereas increases in intramitochondrial Ca2+ paralleled the decreases in DeltaPsiM. (-)-Deprenyl appeared to alter the relationship between intramitochondrial Ca2+ levels and DeltaPsiM, possibly through its reported capacity to increase the synthesis of proteins such as BCL-2.


Assuntos
Apoptose/fisiologia , Proteínas Sanguíneas/farmacologia , Mitocôndrias/metabolismo , Fatores de Crescimento Neural/farmacologia , Fármacos Neuroprotetores/farmacologia , Selegilina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/fisiologia , Cromatina/fisiologia , Citoplasma/metabolismo , Fragmentação do DNA , Radicais Livres/metabolismo , Processamento de Imagem Assistida por Computador , Membranas Intracelulares/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Microscopia de Fluorescência , Mitocôndrias/efeitos dos fármacos , Células PC12 , Peróxidos/metabolismo , Ratos
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