Poly(ADP-ribosyl)ation is a eukaryotic posttranslational modification of proteins that is strongly induced by the presence of DNA strand breaks and plays a role in DNA repair and the recovery of cells from DNA damage. We compared poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) activities in Percoll gradient-purifled, permeabilized mononuclear leukocytes from mammalian species of dfferent maximal life span. Saturating concentrations of a double-stranded octameric oligonucleotide were applied to provide a direct and maximal stimulation of PARP. Our results on 132 individuals from 13 different species yield a strong positive correlation between PARP activity and life span (r = 0.84; P << 0.001), with human cells displaying -5 times the activity of rat cells.Intraspecies comparisons with both rat and human cells from donors ofall age groups revealed some decline ofPARP activity with advancing age, but it was only weakly correlated. No sigicant polymer degradation was detectable under our assay conditions, ruling out any interference by poly(ADPribose) glycohydrolase activity. By Western blot analysis of mononuclear leukocytes from 11 species, using a crave antiserum directed against the extremely well-conserved NADbinding domain, no correlation between the amount of PARP protein and the species' life spans was found, suggesting a greater specific enzyme activity in longer-lived species. We propose that a higher poly(ADP-ribosyl)ation cacit in cells from long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span.Interestingly, Pero et al. (20) We therefore set up a method to provide a direct stimulus for PARP in permeabilized cells-i.e., addition of saturating amounts of a double-stranded oligonucleotide (29 (3,4). DNA break binding leads to an immediate and drastic activation of the catalytic center located in the carboxyl-terminal NADbinding domain; the latter is separated from the DNA-binding domain by a central automodification domain. A large number of studies done in a variety of experimental systems led to the view that poly(ADP-ribosyl)ation plays a role in DNA repair (5) and other cellular responses to DNA damage, such as cell cycle perturbations (6), DNA amplification (7)(8)(9), and malignant transformation (10, 11). Apart from this, poly-(ADP-ribosyl)ation was thought to play a role in DNA replication (12, 13), integration of transfected foreign DNA into the cell genome (14, 15), intrachromosomal homologous recombination (16), differentiation (17, 18), and aging (19-22). In no case, however, have the molecular mechanisms been elucidated so far. MATERIALS AND METHODS

Cisplatin (DDP) is a clinically important antitumor drug that induces the formation of DNA-DNA and DNA-protein crosslinks. We have studied whether poly(ADP-ribosyl)ation, a post-translational modification of nuclear proteins that is drastically increased by the presence of DNA strand breaks and plays a role in DNA repair, is induced following DDP treatment of cell cultures. By using an immunofluorescence technique for the in situ detection of poly(ADP-ribose) in intact cells, we found spotty nuclear signals after DDP treatment of O-342 rat ovarian tumor cells or CV-1 monkey cells, but not in untreated control cells, nor in DDP-treated cells postincubated with the ADP-ribosylation inhibitor 3-aminobenzamide. Our results thus provide direct evidence for an involvement of poly(ADP-ribosyl)ation in the cell's response to DDP treatment and, more generally, illustrate the versatility of this rapid in situ method for the detection of increased poly(ADP-ribosyl)ation in living cells.

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