RESUMO
Two epithelial tumour lines, HeLa and KB, were treated with okadaic acid and calyculin A, specific inhibitors of Ser/Thr phosphatases (PP), esp. PP1 and PP2A. Morphological criteria, analysis of DNA fragmentation and studies of membrane integrity revealed that both agents concentration- and time-dependently induced apoptosis at nanomolar concentrations which in these cells was associated with the stimulation of a transglutaminase activity. Since a non-functional derivative of okadaic acid did not affect cell viability apoptosis was apparently related to the inhibition of PP1 and PP2A. Membrane damage marker activity was delayed by at least 24 h when compared to nuclear alterations.
Assuntos
Apoptose , Fosfoproteínas Fosfatases/fisiologia , Transglutaminases/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Fragmentação do DNA , Relação Dose-Resposta a Droga , Ativação Enzimática , Epitélio/efeitos dos fármacos , Epitélio/enzimologia , Epitélio/fisiologia , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Toxinas Marinhas , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Two structurally different inhibitors of ser/thr phosphatases 1 and 2A, okadaic acid and calyculin A, time- and concentration-dependently stimulated and inhibited cell-specific function (hormone gene expression) in pituitary GH3 cells. The negative effect was associated with the appearance of apoptotic cell death. Nanomolar concentrations of both agents produced the characteristic morphological alterations and a DNA fragmentation ladder. Calyculin A treatment resulted in comparable changes with 10fold lower concentrations than okadaic acid. Observations with derivatives of okadaic acid with no or lower phosphatase inhibitory potency supported the conclusion that apoptosis induction is related to inhibition of ser/thr phosphatases, presumably types 1 and 2A. Membrane damage as measured by lactate dehydrogenase liberation into medium was significantly lower in apoptotic vs. necrotic cells. DNA fragmentation could be reduced by the addition of zinc but not by removal of extracellular calcium with EGTA. Apoptotic changes were reduced by the concomitant activation of protein kinase A by a membrane permeable cAMP analogue. Incubation of cells for 4 months in successively increased concentrations of okadaic acid resulted in a population that proliferated at the initially lethal concentration of 30 nM.