Nj. Donato et M. Perez, TUMOR NECROSIS FACTOR-INDUCED APOPTOSIS STIMULATES P53 ACCUMULATION AND P21WAF1 PROTEOLYSIS IN ME-180 CELLS, The Journal of biological chemistry, 273(9), 1998, pp. 5067-5072
Tumor necrosis factor (TNF)-mediated apoptotic signaling has been char
acterized by activation of specific protease or protein kinase cascade
s that regulate the onset of apoptosis, TNF has also been shown to ind
uce oxidative or genotoxic stress in some cell types, and apoptotic po
tential may be determined by the cellular response to this stress, To
determine the role of genotoxic stress in TNF-mediated apoptosis, we e
xamined cellular accumulation of p53 in TNF-treated ME-180 cells selec
ted for apoptotic sensitivity (ME-180S) or resistance (ME-180R) to TNF
, Although TNF was able to activate receptor-mediated signaling in eit
her cell line, p53 accumulation was measurable only in apoptotically s
ensitive ME-180S cells. TNF-induced changes in p53 levels were detecte
d Ih after treatment, and peak levels were measurable 4-8 h after TNF
exposure. TNF was unable to induce p21WAF1 in either cell line but aff
ected the stability of this protein in apoptotically responsive ME-180
S cells, Evidence of p21WAF1 proteolysis was detected by monitoring th
e appearance of a 16-kDa immunoblottable p21WAF1 fragment, which becam
e detectable 4 h after TNF addition and increased in content before th
e onset of DNA fragmentation (16-24 h), The kinetics of p21WAF1 proteo
lysis closely paralleled those of poly(ADP-ribose) polymerase, suggest
ing cleavage of p21WAF1. by activation of an apoptotic protease, Pretr
eatment of ME-180S cells with the apoptotic protease inhibitor YVAD bl
ocked TNF-induced apoptosis and prevented both poly(ADP-ribose) polyme
rase and p21WAF1 degradation but did not affect p53 induction. These r
esults provide evidence for the early onset of genotoxic stress in cel
ls committed to TNF-mediated apoptosis and for divergence in propagati
on of this signal in non-responsive cells. In addition, TNF-induced p2
1WAF1 proteolysis may be mediated by an apoptotic protease and may con
tribute to the apoptotic process by disrupting p53 signaling, altering
cell cycle inhibition, and limiting cellular recovery from genotoxic
stress.