Biologische Konsequenzen einer Veränderung der zellulären Poly(ADP-Ribose) Polymerase-1 Expression in Nager-Zellen

Poly(ADP-ribosyl)ation is a posttranslational modification of cellular proteins mostly catalysed by poly(ADP-ribose) polymerase-1 (PARP-1) and to a lesser extent by PARP-2. PARP-1 and PARP-2 use NAD+ as substrate in response to cellular exposure to various DNA-damaging agents to form the biopolymer poly(ADP-ribose) (PAR). On the one hand, poly(ADP-ribosyl)ation is a key regulator of genomic stability under conditions of genotoxic stress where PARP-1 plays a major role in DNA repair, transcription regulation and recovery of cells after DNA damage. On the other hand, massive poly(ADP-ribosyl)ation induced by severe acute DNA damage results in rapid depletion of cellular NAD+ and ATP pools, which can lead to cell death.In three sub-projects, this study aimed to explore the role of PARP-1 in DNA repair and cell death using in vitro as well as in vivo models:(i) PARP-1 is involved in a number of pathophysiological conditions such as diabetes, inflammation and stroke, consequently, pharmacological inhibitors of PARP have the potential to elicit beneficial effects in these diseases. In the first part of the present study, a new PARP inhibitor, BYK204165, was examined for inhibition of PAR-synthesis in H2O2-treated 3T3 fibroblasts from Parp-1+/+ and Parp-1-/- mice, where the 100-fold PARP-1 selectivity of the compound was confirmed by its failure to inhibit PARP-2 in both cell lines. The new compound might provide a novel and convenient functional tool toward the assessment of the contribution of PARP-1 and PARP-2 related PAR formation in intact cells, because the enzymatic activity of the two isoforms can be distinguished by use of a selective PARP-1 inhibitor.(ii) Since inhibition of PAR formation generally influences DNA repair mechanisms, the second part of the study explored the consequences of stably overexpressed human PARP-1 (hPARP 1) in Chinese hamster cells (COMF10) on the cytotoxicity induced by alkylating agents (MMS, MNNG) and X-irradiation. Measurements of apoptosis, necrosis, DNA repair and genomic stability were taken as experimental endpoints. Analysis of cell viability after treatment with MMS and MNNG revealed consistently larger fractions of necrotic cells in the COMF10 cells compared to control. Furthermore, DNA repair kinetic measurements after X irradiation of hPARP-1 overexpressing murine lymphoma EL-4 cells, demonstrated acceleration in DNA repair, whereas pharmacological inhibition of PARP by PJ34 delayed and reduced DNA repair capacity.(iii) Finally, it was intended to generate an in vivo system for tissue-specific overexpression of hPARP-1 protein in mice. Therefore, 18 transgenic founder mice were generated by DNA microinjection with a transgene comprising hPARP-1 cDNA under the control of a strong promoter. The transcription is "locked" by a Stop-sequence that can be eliminated in vivo by expression of Cre recombinase as a result of crossing with appropriate transgenic tissue-specific "Cre-deleter" mice (at first T-cell specific). Unexpectedly however, although mRNA transcripts of the hPARP-1 transgene could be found in their offspring, for largely unknown reasons its respective protein expression could not be detected. Its failure on the level of translation possibly could be due to unexpected transcriptional start sites within the transgene. Therefore, an alternative approach should be used for follow-up projects in order to obtain hPARP-1-overexpressing mice.In summary, studies within the work of this thesis contributed to the disposal of a novel and selective PARP-1 inhibitor, which provides a valuable tool to dissect different roles of PARP 1 and PARP-2 in cellular functions (Eltze et al., 2008). Moreover, it was shown that overexpression of hPARP-1 in rodent cells has two important consequences. Its overexpression leads to an increased DNA repair capacity after X-irradiation, but on the other hand also to an increased susceptibility to DNA damage in response to alkylating agents or PARP inhibition, demonstrating the dual role of PARP-1 in mechanisms of DNA repair and cell death (Eltze and Kunzmann et al., submitted). Finally, novel transgenic mice with intended tissue-specific overexpression of hPARP-1 were generated and characterized on a genetic level. Unexpectedly, despite cell culture validation of the expression construct and transgene expression on the mRNA level, no protein expression of the transgene could be detected in these mice for largely unknown reasons. This outcome needs to be considered in future approaches aiming at the generation of hPARP-1 transgenic mice