Cleavage of poly(ADP-ribose) transferase during p53-independent apoptosis in rat liver after treatment with N-nitrosomorpholine and cyproterone acetate

The aim of this work was to study the role of the tumor suppressor p53 and of poly(ADP-ribose) transferase (pADPRT) in the control of hepatocyte apopt osis in two different in vivo models, i.e., during the process of tumor ini tiation by the genotoxin and cytotoxin N-nitrosomorpholine (NNM) and after withdrawal of the hepatomitogen cyproterone acetate (CPA). Treatment with N NM induces apoptosis followed by necrosis and regenerative DNA synthesis. A t the first wave of apoptosis 12 h after NNM application, no p53 expression could be detected by immunohistochemical analysis and immunoblotting. Howe ver, 24 h after treatment, numerous p53-positive hepatocyte nuclei were det ected, whereas hepatocytes in early and later stages of apoptosis were alwa ys negative. Simultaneously with the increased p53 levels, p21 protein was induced. This was accompanied by a block in replicative DNA synthesis, as d etected by proliferating-cell nuclear antigen immunostaining. Concomitantly with the increase in apoptosis, dramatic degradation of the nuclear enzyme pADPRT was observed, as evidenced by immunoblotting and activity blotting. The decrease in pADPRT enzymatic activity observed 12 h after treatment co incided with the greatest extent of pADPRT cleavage. One prominent cleavage product was 64 kDa, suggesting that granzyme B was involved in pADPRT degr adation. In the second in vivo model we used, i.e., withdrawal of treatment with the hepatomitogen CPA, apoptosis of excessive hepatocytes but no necr osis occurs. Again, no induction of p53 expression could be detected in the liver even at the maximum level of apoptosis, whereas a strong correlation between induction of apoptosis and cleavage of pADPRT to a 64-kDa fragment was observed. These results from whole-animal experiments strongly suggest that the induction of apoptosis in rat liver after genotoxic and cytotoxic damage and during regression of hyperplasia is driven by a p53-independent pathway but is accompanied by cleavage of pADPRT. (C) 1999 Wiley-Liss, Inc .