Abstract
Tumor necrosis factor (TNF)-mediated apoptotic signaling has been characterized by activation of specific protease or protein kinase cascades that regulate the onset of apoptosis. TNF has also been shown to induce oxidative or genotoxic stress in some cell types, and apoptotic potential may be determined by the cellular response to this stress. To determine the role of genotoxic stress in TNF-mediated apoptosis, we examined cellular accumulation of p53 in TNF-treated ME-180 cells selected for apoptotic sensitivity (ME-180S) or resistance (ME-180R) to TNF. Although TNF was able to activate receptor-mediated signaling in either cell line, p53 accumulation was measurable only in apoptotically sensitive ME-180S cells. TNF-induced changes in p53 levels were detected 1 h after treatment, and peak levels were measurable 4-8 h after TNF exposure. TNF was unable to induce p21WAF1 in either cell line but affected the stability of this protein in apoptotically responsive ME-180S cells. Evidence of p21WAF1 proteolysis was detected by monitoring the appearance of a 16-kDa immunoblottable p21WAF1 fragment, which became detectable 4 h after TNF addition and increased in content before the onset of DNA fragmentation (16-24 h). The kinetics of p21WAF1 proteolysis closely paralleled those of poly(ADP-ribose) polymerase, suggesting cleavage of p21WAF1 by activation of an apoptotic protease. Pretreatment of ME-180S cells with the apoptotic protease inhibitor YVAD blocked TNF-induced apoptosis and prevented both poly(ADP-ribose) polymerase and p21WAF1 degradation but did not affect p53 induction. These results provide evidence for the early onset of genotoxic stress in cells committed to TNF-mediated apoptosis and for divergence in propagation of this signal in non-responsive cells. In addition, TNF-induced p21WAF1 proteolysis may be mediated by an apoptotic protease and may contribute to the apoptotic process by disrupting p53 signaling, altering cell cycle inhibition, and limiting cellular recovery from genotoxic stress.
Highlights
The process of cell death or apoptosis has gained wide recognition as an important mediator of organismal development and pathology
The distinctions in specific genotoxic stress signals were investigated in ME-180 cells selected for sensitivity or resistance to Tumor necrosis factor (TNF)-induced apoptosis
Mutations in p53 and general defects in apoptosis in ME-180R cells do not appear to account for the loss of sensitivity to TNF since other apoptotic stimuli were able to induce equivalent or greater levels of apoptosis in ME-180R and ME-180 cells selected for apoptotic sensitivity (ME-180S) cells [45]
Summary
The process of cell death or apoptosis has gained wide recognition as an important mediator of organismal development and pathology. The results suggest that TNF activated stress signaling cascades (JNK, NF-B) in both cell types, early genotoxic stress, as measured by p53 accumulation, was detected only in apoptotically sensitive cells. Twenty-four hours after TNF addition and after the onset of DNA fragmentation, p21WAF1 protein was not detectable by immunoblotting apoptotic cell lysates.
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