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(-)/(-)A11 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 A11 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.
Aoufouchi
S
S
Laboratory of Molecular Biology, Medical Research Council, Hills Road, Cambridge, CB2 2QH, UK. aoufouchi@meckerifr
Yélamos
J
J
Milstein
C
C
D000478:Alkylation / Q000187:drug effects; D017304:Annexin A5 / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts; D017353:Gene Deletion; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008770:Methylnitrosourea / Q000494:pharmacology; D009154:Mutation; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection
D017304:Annexin A5; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D008769:Methylnitronitrosoguanidine; D008770:Methylnitrosourea; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine