Overexpressed poly(ADP-ribose) polymerase delays the release of rat cells from p53-mediated G(1) checkpoint

We have previously reported that in cells ectopically expressing temperatur e-sensitive p53(135val) mutant, p53 formed tight complexes with poly(ADP-ri bose) polymerase (PARP). At elevated temperatures, p53(135val) protein, ado pting the mutant phenotype, was localized in the cytoplasm and sequestered the endogenous PARP. To prove whether an excess of p53(135val) protein led to this unusual intracellular distribution of PARP, we have established cel l lines overexpressing p53(135val) + c-Ha-ras alone or in combination with PARP. interestingly, immunostaining revealed that PARP is sequestered in th e cytoplasm by mutant p53 in cells overexpressing both proteins. Simultaneo us overexpression of PARE had no effect on temperature-dependent cell proli feration and only negligibly affected the kinetics of p53-mediated G(1) arr est. However, if the cells were completely growth arrested at 32 degreesC a nd then shifted up to 37 degreesC, coexpressed PARP dramatically delayed th e reentry of transformed cells into the cell cycle. Even after 72 h at 37 d egreesC the proportion of S-phase cells was reduced to 20% compared to thos e expressing only p53(135val) + c-Ha-ras. The coexpressed PARP stabilized w t p53 protein and its enzymatic activity was necessary for stabilization. ( C) 2000 Wiley-Liss, Inc.