Einfluß der Poly(ADP-Ribosyl)ierung auf die genomische Stabilität nach genotoxischer Behandlung in Hamsterzell-Transfektanten

Abstract

The formation of poly(ADP-ribose) (pADPr) from nicotinamide-adenine dinucleotide (NAD+) is a reaction catalysed in the cell by a family of enzymes called poly(ADP-ribose)-polymerases (PARPs). Among these, poly(ADP-ribose)-polymerase-1 (PARP-1, EC 2.4.2.30) is responsible for the formation of about 90% of total polymer induced by DNA strand breaks, which are potent activators of this enzyme. The product, pADPr, is transferred to acceptors, mainly PARP-1 itself and other nuclear proteins. PARP-1 has been shown to be neccessary for maintaining genome stability, via interaction and complex formation with proteins of the DNA repair machinery. In this study a cell line was used carrying inducible transgenes for either full length PARP-1 or its DNA binding domain (DBD), the latter being known to inhibit the binding of endogenous PARP-1 to DNA strand breaks. These cells served as a system for up- or down regulation of PARP-1 activity, in order to investigate the influence of the enzyme for genome stability after DNA damage induction by the alkylating agent MNNG or bleomycin. Integrity of the genome was assessed by scoring micronucleus formation. I was able to show a significant increase of micronucleus formation in DBD-expressing cells after treatment with bleomycin. Vice versa the over expression of PARP-1 led to a drastic decrease of micronuclei formation after DNA damage induction. A similar increase or decrease in genomic stability was also shown in previous papers by corresponding chamges in sister-chromatide exchange frequency. Additionally, cells lacking PARP-1 activity were more prone to undergo apoptosis after DNA-damaging treatment, which is an indication for impaired DNA repair capacity. Consistent with other reports was also the finding that overexpression of PARP-1 in COMF10 cells did not lead to protection from cytotoxicity, but actually increased necrotic cell death at high MNNG concentrations. Although the molecular mechanism of PARP-1 action has not yet been elucidated there is clear evidence for a contribution to base-excision-repair (BER) and a regulatory role in homologous recombination. Overall these results suggest that PARP-1 plays an important role for DNA damage recovery in the cell. A beneficial, cytoprotective effect of PARP-1 activity has been shown here, manifested as reduced micronuclei formation and reduced apoptosis.