Zinc inhibits apoptosis upstream of ICE/CED-3 proteases rather than at the level of an endonuclease.

Apoptosis is commonly associated with DNA digestion, but it remains controversial as to which endonuclease is involved. The ability of zinc to inhibit DNA digestion in intact cells, and inhibit a Ca2+/Mg2+-dependent endonuclease in cell lysates, has been used frequently to suggest this is the endonuclease involved. However, zinc has many other effects on cells, and here it is shown that zinc also prevents many upstream events in apoptosis. These studies were performed in human ML-1 cells following incubation with etoposide. During apoptosis, these cells undergo intracellular acidification, increased accumulation of Hoechst 33342, DNA digestion and chromatin condensation. Zinc inhibited all of these events. An upstream event in apoptosis is activation of ICE/CED-3 proteases which is commonly observed as proteolysis of a substrate protein, poly(ADP-ribose) polymerase (PARP). The ICE/CED-3 proteases are themselves activated by proteolysis, and this was detected here by cleavage of one family member CPP32. Zinc prevented cleavage of both CPP32 and PARP. We recently demonstrated that dephosphorylation of the retinoblastoma susceptibility protein Rb was a marker of an event even further upstream in apoptosis; zinc was also found to inhibit Rb dephosphorylation. Therefore, zinc must protect cells at a very early step in the apoptotic pathway, and not as a direct inhibitor of an endonuclease.

The temporal relationship between protein phosphatase, ICE/CED-3 proteases, intracellular acidification, and DNA fragmentation in apoptosis.

Apoptosis occurs during development and tissue homeostasis, and under conditions of physical and chemical stress. During apoptosis, cells digest their DNA, decrease intracellular pH, shrink, exhibit protein phosphatase activity, and activate members of the ICE/CED-3 family of proteases. This protease activity is identified by cleavage of poly(ADP-ribose) polymerase (PARP). Phosphatase activity during apoptosis is observed as dephosphorylation of the retinoblastoma susceptibility protein (Rb). Serine/threonine phosphatase inhibitors can prevent dephosphorylation of Rb and apoptosis, suggesting that Rb dephosphorylation is an indication of a critical regulator of apoptosis. The experiments described here were designed to establish the temporal relationship between these events. Apoptosis was induced in human ML-1 cells by the topoisomerase inhibitor etoposide. An inhibitor of the ICE/CED-3 protease family, z-VAD-fluoromethylketone (FMK), showed concentration-dependent protection from PARP cleavage, intracellular acidification, DNA digestion, early changes in membrane permeability, and cell shrinkage, thereby placing all of these events downstream of the ICE/CED-3 protease action. However, z-VAD-FMK did not prevent the dephosphorylation of Rb, placing this change upstream of the protease. These results suggest that the imbalance between protein phosphatase and kinase that is responsible for the dephosphorylation of Rb is also responsible for the activation of ICE/CED-3 proteases, which in turn is responsible for all the other events associated with apoptosis.

The involvement of protein phosphatases in the activation of ICE/CED-3 protease, intracellular acidification, DNA digestion, and apoptosis.

Many events in apoptosis have been identified but their temporal relationships remain obscure. Apoptosis in human ML-1 cells induced by etoposide is characterized by intracellular acidification, enhanced Hoechst 33342 fluorescence, DNA digestion, chromatin condensation, and proteolysis of poly(ADP-ribose) polymerase. This proteolysis is a marker for the action of ICE/CED-3 proteases, which are critical activators of apoptosis. We observed that three serine/threonine protein phosphatase inhibitors, okadaic acid, calyculin A, and cantharidin, prevented all of these apoptotic characteristics. To determine which protein phosphatase was involved, we investigated the dephosphorylation of the retinoblastoma susceptibility protein Rb, a substrate for protein phosphatase 1 but not protein phosphatase 2A. Rb was dephosphorylated during apoptosis, and each inhibitor prevented this dephosphorylation at the same concentrations that prevented apoptosis. No increase in protein phosphatase 1 activity was observed in apoptotic cells suggesting that dephosphorylation of Rb may result from loss of Rb kinase activity in the presence of a constant level of protein phosphatase activity. Long term inhibition of protein phosphatase 1 (>8 h) also led to the appearance of dephosphorylated Rb, cleavage of poly(ADP-ribose) polymerase and apoptosis, suggesting these events are not solely dependent upon protein phosphatase 1. Rb dephosphorylation was also observed in several other models of apoptosis. Hence, an imbalance between protein phosphatase 1 and Rb kinase may be a common means to activate ICE/CED-3 proteases resulting in the subsequent events of apoptosis.

Oxidant-sensitive and phosphorylation-dependent activation of NF-kappa B and AP-1 in endothelial cells.

Relatively low concentrations of reactive oxygen cause reversible alterations of endothelial cell signal transduction and gene transcription. The hypothesis that low levels of oxidant stress activate retention of trans-acting proteins in the nucleus was investigated by determining time and dose requirements for oxidant-stimulated nuclear protein binding to consensus DNA sequences for nuclear factor (NF)-kappa B or activator protein 1 (AP-1). Nuclear proteins were extracted from low passage porcine aortic endothelial cells 15 min to 24 h after addition of increasing concentrations of H2O2. Electrophoretic mobility shift assays demonstrated that protein binding to NF-kappa B and AP-1 sequences increases over 1-2 h after stress relative to time-matched controls and resolves by 24 h. The selective protein kinase C inhibitor, calphostin C, prevents approximately 30% of this increase. Inhibition of tyrosine kinase activity by herbimycin A (5 microM) completely inhibits the response to H2O2. Exposure of intact cells to H2O2 increases substrate phosphorylation in pp60src immunoprecipitates. The activity of pp60src in immunoprecipitates from control cells or of recombinant pp60src increases after in vitro addition of H2O2. H2O2-stimulated pp60src activity is reduced by pretreatment of the enzyme preparation with N-acetylcysteine. These data indicate that oxidants increase nuclear levels of trans-acting factors in endothelial cells and that these increases require oxidant-sensitive changes in both tyrosine and serine/threonine phosphorylations.

Interactions between hydroxocobalamin and nitric oxide (NO): evidence for a redox reaction between NO and reduced cobalamin and reversible NO binding to oxidized cobalamin.

Interactions of nitric oxide (NO) with various cobalamin species have been examined, apparently for the first time, with both absorption and electron paramagnetic resonance spectroscopy. Only slight shifts in the absorption spectrum of hydroxocobalamin, B12a [Cb(III)], were produced by NO, but dramatic changes in the spectrum of B12r [Cb(III)] were found on addition of NO. The addition of NO shifted the spectrum of Cb(II) to one very similar to that of Cb(III), indicating the oxidation of Cb(II). The addition of NO to Cb(III) resulted in a novel, weak and previously undescribed electron paramagnetic resonance signal. Although it has not been fully characterized, this appears to represent a reversible complex in which NO is liganded to the Cb(III). When NO was added to Cb(II), its strong electron paramagnetic resonance spectrum was replaced by that of this novel species, consistent with oxidation of Cb(II) by NO and then binding of additional NO by the resulting Cb(III). Porcine, aortic endothelial cells were able to partially reduce Cb(III), and release to the supernatant a previously characterized superoxide cobalt(III) complex, but some Cb(II) remained with the cell fraction. These reactions of Cb species could play a role in altering intracellular and intratissue levels of NO.