RESUMO
The cell surface molecules controlling apoptosis in cortical neurons are largely unknown. A monoclonal antibody was derived that induces cultured neocortical neurons to undergo apoptosis. A Fab fragment of the antibody, however, lacked the ability to induce cell death. The antigen was purified, and characterized by compositional analysis, fast atom bombardment (FAB) mass spectrometry, sequential exoglycosidase treatments, methylation analysis, and (1)H-nuclear magnetic resonance spectroscopy, proving to be isoglobotetraosylceramide (IsoGb4). IsoGb4 has been shown previously to be a metastasis marker, antibodies against which block metastases in a mammary adenocarcinoma model (S. A. Carlsen et al., Cancer Res., 53: 2906-2911, 1993). Addition of the purified antigen to cells lacking this glycolipid demonstrated that it is capable of functioning as a portable apoptosis-transducing molecule. Intracellular ceramide levels were increased after the treatment with the apoptosis-inducing antibody, but the membrane sphingomyelin level remained unchanged. Fumonisin B1 inhibited both the ceramide increase and the apoptosis induced via IsoGb4, which indicated that the ceramide synthase pathway is likely to be involved in apoptosis induction by IsoGb4.
Assuntos
Anticorpos Monoclonais/metabolismo , Antígenos de Superfície/metabolismo , Apoptose/fisiologia , Globosídeos/metabolismo , Neurônios/citologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Antígenos de Superfície/imunologia , Antígenos de Superfície/isolamento & purificação , Apoptose/imunologia , Sequência de Carboidratos , Transformação Celular Neoplásica , Globosídeos/imunologia , Globosídeos/isolamento & purificação , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Neurônios/imunologia , Neurônios/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Three of the predominant features of apoptosis are internucleosomal DNA fragmentation, plasma membrane bleb formation, and retraction of cell processes. We demonstrate that actin is a substrate for the proapoptotic cysteine protease interleukin 1beta-converting enzyme. Actin cleaved by interleukin 1beta-converting enzyme can neither inhibit DNase I nor polymerize to its filamentous form as effectively as intact actin. These findings suggest a mechanism for the coordination of the proteolytic, endonucleolytic, and morphogenetic aspects of apoptosis.
Assuntos
Actinas/metabolismo , Apoptose , Cisteína Endopeptidases/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/antagonistas & inibidores , Animais , Caspase 1 , Humanos , Interleucina-1/metabolismo , Células PC12 , Mapeamento de Peptídeos , Ligação Proteica , Ratos , Especificidade por SubstratoRESUMO
Glutamate has been shown, at high concentrations (5-10 mM), to lead to death in cells of the pheochromocytoma cell line PC12. We report that similar concentrations of glutamate also kill immortalized central neural cell lines, and that the expression of the proto-oncogene bcl-2 in these cell lines blocks glutamate neurotoxicity. Potassium chloride (25 mM) also protects a cerebellar neuronal cell line, but not PC12 cells, from glutamate toxicity.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Glutamatos/toxicidade , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Transfecção , Animais , Neoplasias Encefálicas , Células CHO , Linhagem Celular Transformada , Cerebelo , Cricetinae , Antagonistas de Aminoácidos Excitatórios , Ácido Glutâmico , Humanos , Camundongos , Neurônios/citologia , Neurotoxinas/antagonistas & inibidores , Células PC12 , Cloreto de Potássio/farmacologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2 , RatosRESUMO
Nerve growth factor (NGF) binding to cellular receptors is required for the survival of some neural cells. In contrast to TrkA, the high-affinity NGF receptor that transduces NGF signals for survival and differentiation, the function of the low-affinity NGF receptor, p75NGFR, remains uncertain. Expression of p75NGFR induced neural cell death constitutively when p75NGFR was unbound; binding by NGF or monoclonal antibody, however, inhibited cell death induced by p75NGFR. Thus, expression of p75NGFR may explain the dependence of some neural cells on NGF for survival. These findings also suggest that p75NGFR has some functional similarities to other members of a superfamily of receptors that include tumor necrosis factor receptors, Fas (Apo-1), and CD40.
Assuntos
Apoptose , Fatores de Crescimento Neural/metabolismo , Neurônios/citologia , Receptores de Fator de Crescimento Neural/fisiologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro , Fatores de Crescimento Neural/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Receptores de Fator de Crescimento Neural/metabolismo , TransfecçãoRESUMO
The protooncogene bcl-2, which has been implicated in B-cell lymphoma development, inhibits apoptosis due to growth factor withdrawal in some, but not all, hematopoietic cells. Recently we found that bcl-2 also inhibits apoptosis in PC12 pheochromocytoma cells. We now report that bcl-2 inhibits the death of a central neural cell line due to serum and growth factor withdrawal, the calcium ionophore A23187, glucose withdrawal, membrane peroxidation, and, in some cases, free radical-induced damage. This broad range of protective effects of BCL-2 protein suggests that BCL-2 may interact with a central step in neural cell death. Measurements of intracellular free calcium suggest that BCL-2 alters the transduction of neural death signals at a point distal to the rise in intracellular free calcium.
Assuntos
Apoptose , Morte Celular , Neurônios/citologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Cálcio/fisiologia , Expressão Gênica , Técnicas In Vitro , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
During development, many neuronal populations undergo a process of normal, programmed cell death, or apoptosis. Trophic factors regulate this process, but the mechanism by which they suppress apoptosis remains unclear. In the immune system, recent studies have implicated the protooncogene bcl-2 in the lymphocyte survival response to growth factors. To determine whether a similar survival pathway exists in a neuroendocrine cell type, we have expressed bcl-2 in the rat pheochromocytoma PC12 cell line and found that it abrogates the requirement for stimulation by growth factors to survive. bcl-2 expression also substantially delays the onset of injury by the calcium ionophore A23187.