trans-dominant inhibition of poly(ADP-ribosyl)ation sensitizes cells against gamma-irradiation and N-methyl-N'-nitro-N-nitrosoguanidine but does not limit DNA replication of a polyomavirus replicon.
Poly(ADP-ribosyl)ation is a posttranslational modification of nuclear proteins catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30), with NAD+ serving as the substrate. PARP is strongly activated upon recognition of DNA strand breaks by its DNA-binding domain. Experiments with low-molecular-weight inhibitors of PARP have led to the view that PARP activity plays a role in DNA repair and possibly also in DNA replication, cell proliferation, and differentiation. Accumulating evidence for nonspecific inhibitor effects prompted us to develop a molecular genetic system to inhibit PARP in living cells, i.e., to overexpress selectively the DNA-binding domain of PARP as a dominant negative mutant. Here we report on a cell culture system which allows inducible, high-level expression of the DNA-binding domain. Induction of this domain leads to about 90% reduction of poly(ADP-ribose) accumulation after gamma-irradiation and sensitizes cells to the cytotoxic effect of gamma-irradiation and of N-methyl-N'-nitro-N-nitrosoguanidine. In contrast, induction does not affect normal cellular proliferation or the replication of a transfected polyomavirus replicon. Thus, trans-dominant inhibition of the poly(ADP-ribose) accumulation occurring after gamma-irradiation or N-methyl-N'-nitro-N-nitrosoguanidine is specifically associated with a disturbance of the cellular recovery from the inflicted damage.
Küpper
J H
JH
Abteilung 0610, Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Müller
M
M
Jacobson
M K
MK
Tatsumi-Miyajima
J
J
Coyle
D L
DL
Jacobson
E L
EL
Bürkle
A
A
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002455:Cell Division / Q000187:drug effects / Q000528:radiation effects; D002461:Cell Line, Transformed; D006224:Cricetinae; D003412:Cricetulus; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005720:Gamma Rays; D018014:Gene Transfer Techniques; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D011120:Polyomavirus / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D012093:Replicon; D014779:Virus Replication / Q000187:drug effects / Q000528:radiation effects
D004268:DNA-Binding Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D008769:Methylnitronitrosoguanidine; D011065:Poly(ADP-ribose) Polymerases