RESUMO
Human promyelocytic leukaemia cells (HL-60) differentiate into neutrophil-like cells that die spontaneously by apoptosis when treated with retinoic acid (RA). Inhibitors of apoptosis proteins (IAP) bind to and inhibit caspases 3, 7, and 9 activity and the induction of apoptosis. In this study, we demonstrate that undifferentiated HL-60 cells express IAP. During their differentiation, IAP expression is decreased at the mRNA and protein levels. In addition, we show that there is a corresponding increase in the expression and functional activity of active caspases 3 and 9. This activity was associated with the cleavage of XIAP, NAIP, and cIAP-2. Most importantly, we demonstrate that blocking caspase activity does not alter the decrease in IAP protein expression during differentiation but prevents caspase activation, IAP cleavage, and the induction of apoptosis. This result shows that the loss of IAP expression is independent of the induction of apoptosis and is solely related to the differentiation process. However, IAP cleavage is caspase-dependent. Terminal differentiation results in an altered apoptotic phenotype that is associated with the induction of HL-60 cell apoptosis.
Assuntos
Caspases/metabolismo , Células HL-60/fisiologia , Proteínas do Tecido Nervoso/biossíntese , Biossíntese de Proteínas , Proteínas , Apoptose , Diferenciação Celular/fisiologia , Ativação Enzimática , Células HL-60/citologia , Humanos , Proteínas Inibidoras de Apoptose , Proteína Inibidora de Apoptose Neuronal , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo XRESUMO
Using synapses which form between somata of Helisoma neurons in cell culture we have studied the presynaptic regulation of synaptic transmission. GTP-binding proteins play important roles in regulating synaptic transmission through their actions on calcium currents, potassium currents and secretory apparatus. Heterotrimeric G proteins continuously regulate the amount of transmitter released at the synapse. By interacting with the arachidonic acid second messenger system they modulate potassium channels, and could potentially control the secretory apparatus. Perturbations of the rab protein system did not affect action potential-evoked transmission, but did control the frequency of miniature inhibitory postsynaptic currents. This is consistent with the involvement of this type of GTP-binding protein in the control of secretory apparatus, but suggests that rab proteins are not used to regulate the amount of transmitter released at the synapse. Using the Helisoma cellular system which permits direct access to the presynaptic site of transmitter release we are going on to study further the role of arachidonic acid, Go, Gi and rab proteins on the regulation of the secretory apparatus.
Assuntos
Ácido Araquidônico/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Transmissão Sináptica/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Cálcio/farmacologia , FMRFamida , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Canais Iônicos/efeitos dos fármacos , Dados de Sequência Molecular , Neuropeptídeos/farmacologia , Neurotransmissores/farmacologia , Sondas de Oligonucleotídeos/genética , Mapeamento de Peptídeos , Caramujos , Sinapses/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
HL-60 cell differentiation into neutrophil like cells is associated with their induction of apoptosis. We investigated the cellular events that occur pre and post mitochondrial permeability transition to determine the role of the mitochondria in the induction of differentiation induced apoptosis. Pro-apoptotic Bax was translocated to and cleaved at the mitochondrial membrane in addition to t-Bid activation. These processes contributed to mitochondrial membrane disruption and the release of cytochrome c and Smac/DIABLO. The release of cytochrome c was caspase independent, as the caspase inhibitor Z-VAD.fmk, which inhibited apoptosis, did not block the release of cytochrome c. In contrast, the release of Smac/DIABLO was partially inhibited by caspase inhibition indicating differential release pathways for these mitochondrial pro-apoptotic factors. In addition to caspase inhibition we assessed the effects of the Bcl-2 anti-apoptotic family on differentiation induced apoptosis. BH4-Bcl-xl-TAT recombinant protein did not delay apoptosis, but did block the release of cytochrome c and Smac/DIABLO. Bcl-2 over-expression also inhibited differentiation induced apoptosis but was associated with the inhibition of the differentiation process. Differentiation mediated mitochondrial release of cytochrome c and Smac/DIABLO, may not trigger the induction of apoptosis, as BH4-Bclxl-TAT blocks the release of pro-apoptotic factors from the mitochondria, but does not prevent apoptosis.