Inhibition of apoptosis of a PARP-/- cell line transfected with PARP DNA-binding domain mutants

We have investigated the possibility of the involvement of PARP in apoptosis, independently of its enzymatic activity. We thus transfected PARP(-/-) All cells with a DNA construct encoding the PARP DNA-binding domain (DBD) fragment or mutants DBDbd(-), defective in DNA binding to DNA strand breaks, and DBDcl(-), resistant to caspase-3 cleavage. We found that in the absence of PARP, while expression of DBD has only a marginal effect, expression of the mutants strongly inhibits the apoptosis induced by staurosporine, as measured by the binding of annexin V. Moreover, the mutants, but not DBD, inhibit the cleavage of DNA PKcs, suggesting inhibition of activation of caspase-3. In addition, the mutant transfectants are fractionally less susceptible to low doses of an alkylating agent than the DBD transfectant or the original All Line. The results suggest that the DBD fragment of PARP, apart from its classical role of nick detection and DNA binding, participates in complexes involved in upstream events leading to activation of the caspase cascade. (C) 1999 Academic Press.