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

The Automated FADU-Assay, a Potential High-Throughput In Vitro Method for Early Screening of DNA Breakage

Genotoxicity tests are essential to identify compounds that have a potential to compromise not only the environment but also human and animal health, including compounds that increase the risk of cancer. At present, no single test is capable of detecting all types of genotoxic effects; therefore a battery of in vitro and, if positive, in vivo tests is necessary to determine the genotoxicity of a substance. However, the respective specificities of current assays have been criticized for their high percentage of false positive results. We recently published an automated version of the “Fluorimetric detection of Alkaline DNA Unwinding” (FADU) assay for measuring DNA strand breaks in human peripheral blood mononuclear cells or in cell lines. Using this new technology we show detection of DNA strand breaks in cells treated with several compounds known to induce DNA strand breaks by various mechanisms. We also tested toxic compounds that were not expected to induce DNA strand breaks; these were negative in the assay as expected. Finally, we included zinc oxide nanoparticles of high production volume to explore further fields of potential FADU applications. The main advantages of this assay are high reproducibility, easy handling, lack of operator bias, high-throughput, speed, and low cost

International ring trial of the epidermal equivalent sensitizer potency assay : reproducibility and predictive-capacity

This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50% cytotoxicity (EE-EC50) or the 2-fold increase in IL-1α (IL-1α2x). Four laboratories received 13 coded sensitizers. Reproducible results were obtained in each laboratory. A binary prediction model, EC50≥7 mg/ml=weak to moderate sensitizer and EC50<7 mg/ml=strong to extreme sensitizer had an accuracy of 77%. A superior EE (EC50 and IL-1α2x) correlation was observed with human in vivo DSA05 data compared to LLNA-EC3 data. Human in vivo NOEL and LLNA-EC3 data correlated to a similar extent to in vitro EE data. Our results indicate that this easily transferable EE potency assay is suitable for testing chemical allergens of unknown potencies and may now be ready for further validation, providing complementary potency information to other assays already undergoing validation for assessing skin sensitization potential

Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency

At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers according to their potency, without the use of animals. Within the Sens-it-iv EU Frame Programme 6 funded Integrated Project (LSHB-CT-2005-018681), a number of in vitro, human cell based assays were developed which, when optimized and used in an integrated testing strategy, may be able to distinguish sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay. The aim of this study is to show the transferability of the two-tiered approach with training chemicals: 3 sensitizers (DNCB, resorcinol, pPD) and 1 non sensitizer (lactic acid) in tier 1 and 2 sensitizers with different potency in tier 2 (DNCB; extreme and resorcinol; moderate). The chemicals were tested in a non-coded fashion. Here we describe the transferability to naïve laboratories, the establishment of the standard operating procedure, critical points, acceptance criteria and project management. Both assays were successfully transferred to laboratories that had not performed the assays previously. The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization. © 2012 Elsevier Ltd

EGFR activity addiction facilitates anti-ERBB based combination treatment of squamous bladder cancer

Recent findings suggested a benefit of anti-EGFR therapy for basal-like muscle-invasive bladder cancer (MIBC). However, the impact on bladder cancer with substantial squamous differentiation (Sq-BLCA) and especially pure squamous cell carcinoma (SCC) remains unknown. Therefore, we comprehensively characterized pure and mixed Sq-BLCA (n = 125) on genetic and protein expression level, and performed functional pathway and drug-response analyses with cell line models and isolated primary SCC (p-SCC) cells of the human urinary bladder. We identified abundant EGFR expression in 95% of Sq-BLCA without evidence for activatingEGFRmutations. Both SCaBER and p-SCC cells were sensitive to EGFR tyrosine kinase inhibitors (TKIs: erlotinib and gefitinib). Combined treatment with anti-EGFR TKIs and varying chemotherapeutics led to a concentration-dependent synergism in SCC cells according to the Chou-Talalay method. In addition, the siRNA knockdown of EGFR impaired SCaBER viability suggesting a putative "Achilles heel" of Sq-BLCA. The observed effects seem Sq-BLCA-specific since non-basal urothelial cancer cells were characterized by poor TKI sensitivity associated with a short-term feedback response potentially attenuating anti-tumor activity. Hence, our findings give further insights into a crucial, Sq-BLCA-specific role of the ERBB signaling pathway proposing improved effectiveness of anti-EGFR based regimens in combination with chemotherapeutics in squamous bladder cancers with wild-type EGFR-overexpression