Analysis of Poly(ADP-ribosyl)ation Metabolism

4.2.2.1 Maximum Poly(ADP-ribosyl)ation Capacity in Permeabilized ES Cells

To investigate if ectopic hPARP-1 expression influences maximum poly(ADP-ribosyl)ation capacity in hPARP-1 ES cells, a recently published immunofluorescence-based flow cytometric technique was employed (Kunzmann et al. 2006). This approach uses permeabilized cells that were incubated in a reaction buffer containing high concentrations of PARP-1‟s substrate NAD⁺ (500 µM) in combination with double-stranded a „activator oligonucleotide‟ to trigger DNA binding of PARP-1 and to stimulate its activity to the maximum. Subsequently, poly(ADP-ribose) was immunostained and immunofluorescence signal intensities were analyzed by flow cytometry. Interestingly, maximum poly(ADP-ribosyl)ation capacities were equal or slightly lower in 1-expressing cells compared to cells showing no ectopic expression of hPARP-1 (wild-type and clone #hPARP-15hPARP-1; Figure 4.hPARP-19).

Figure 4.19. Determination of maximum PARP activity in wild-type and hPARP-1 ES cells.

Flow cytometric measurement of maximum poly(ADP-ribosyl)ation capacity in wild-type (wt) and hemizygous hPARP-1 (#113, #113NeoR-, #225, #225NeoR-) ES cells. Clone #151 exhibited no ectopic expression of hPARP-1 (Figure 4.15) and served as a control for G418-supplemented ES cell culture conditions. Using a reaction buffer containing 500 µM NAD⁺ and double-stranded „activator oligonucleotides‟, maximum PARP-1 activity was induced in permeabilized ES cells for 10 min and thereafter cells were stained for poly(ADP-ribose) using the primary antibody 10H and the fluorophor-labeled secondary antibody AlexaFluor488. Data are from 3 to 5 independent biological experiments each performed in technical triplicates, except for #113NeoR- (two biological experiments). Potential statistical significance was analyzed by Student‟s t test. Means ± SEM. NeoR- indicates lines with excised NeoR cassette.

4.2.2.2 Poly(ADP-ribosyl)ation Capacity in Living ES Cells

The effect of hPARP-1 expression on the cellular poly(ADP-ribosyl)ation response upon genotoxic stimuli was analyzed in intact cells. For this purpose, wild-type and hPARP-1 ES cells were X-irradiated with increasing radiation doses to induce DNA damage. Subsequent poly(ADP-ribose) formation was detected by immunofluorescence analysis. Figure 4.20 shows

poly(ADP-ribose) formation in wild-type ES cells, but also mutant ES cell clones (#113, #225,

#225NeoR-) responded to X-irradiation with formation of poly(ADP-ribose) in a dose-dependent manner.

No significant differences in poly(ADP-ribose) formation between hPARP-1-expressing (clone #113) and non-hPARP-1-expressing ES cells (wild-type and clone #151) were detected with radiation doses less than 5 Gy (Figure 4.21). However, significant differences became obvious in the case of radiation doses of 5 and 15 Gy, although with different effects: In the case of irradiation with 5 Gy, poly(ADP-ribose) formation was 27.7% lower in hPARP-1-expressing cells compared to wild-type cells. In contrast, in the case of irradiation with 15 Gy, poly(ADP-ribose) formation was 43% higher in hPARP-1-expressing cells compared to wild-type cells (Figure 4.21). Similar results were obtained for ES cells of clone #225. They showed a 6.6%

decrease in the case of irradiation with 5 Gy and a 29% increase in the case of irradiation with 15 Gy compared to wild-type ES cells (data not shown).

Figure 4.20. Poly(ADP-ribose) antibody 10H and the fluorophor-labeled secondary antibody

Figure 4.21. Poly(ADP-ribose) levels in wild-type and hPARP-1 ES cells upon X-irradiation.

Immunofluorescence analysis showing wild-type (wt) and hPARP-1 (#113) ES cells that were X-irradiated as indicated and after a 5-min reaction period stained for poly(ADP-ribose) formation using the primary antibody 10H and the fluorophor-labeled secondary antibody AlexaFluor488 (FITC channel). Nuclei were counterstained by Hoechst DNA staining (Hoechst channel). Clone #151 exhibited no ectopic expression of hPARP-1 (Figure 4.15) and served as a control for G418-supplemented ES cell culture conditions. Identical exposure times were used in the case of each irradiation dose. Fluorescence intensities (mean gray values) were quantified from 16-41 nuclei from three to five randomly chosen images. Statistical significance was analyzed by Student‟s t test. Means ± SEM.

Original magnification ×630. Red bars indicate 10 µm.

Figure 4.22. NAD⁺ levels in wild-type and hPARP-1 ES cells and splenocytes.

A. Basal NAD⁺ levels and NAD⁺ levels after X-irradiation with 25 Gy and subsequent 10-min reaction period were determined in wild-type (wt) and hemizygous hPARP-1 (#113, #113NeoR-, #225, #225NeoR-) ES cells using an NAD⁺ cycling assay. Data are from three independent biological experiments each performed in technical triplicates. B. Same experimental procedure as described in A, but with freshly isolated splenocytes from wild-type, hemizygous mutant (1×hPARP-1), and homozygous mutant (2×hPARP-1) mice of line #225NeoR-. Experiments were performed in technical triplicates. Statistical significance was analyzed by Student‟s t test. Means ± SEM.

Conc. indicates concentration; NeoR-, lines with excised NeoR cassette.

4.2.2.3 NAD⁺ Levels in ES Cells and Primary Splenocytes

NAD⁺ serves as the substrate for PARP-1‟s enzymatic activity (Bürkle 2005). To study the effects of the ectopic hPARP-1 expression on NAD⁺ metabolism, intracellular NAD⁺ concentrations were determined in vitro and ex vivo using a NAD⁺ cycling assay (Jacobson and Jacobson 1976). NAD⁺ concentration of wild-type ES cells was determined to be 411 µM ± 58 µM. In agreement with higher poly(ADP-ribosyl)ation capacity of the human enzyme, basal NAD⁺ levels of hPARP-1-expressing ES cells were more than 20% lower

compared to wild-type ES cells (Figure 4.22 A). X-irradiation of ES cells with a dose of 25 Gy led to a reduction in NAD⁺ levels of 10 to 20% in both wild-type and mutant ES cells.

Considerably lower NAD⁺ concentrations were determined ex vivo in freshly isolated splenocytes (47 µM ± 5 µM). Consequently, in comparison with ES cells the relative decrease in NAD⁺ levels after X-irradiation of splenocytes with 25 Gy was more pronounced: ex vivo NAD⁺ levels of splenocytes dropped by more than 60%. Moreover, ex vivo experiments demonstrated no reduction in NAD⁺ levels in mutant compared to wild-type splenocytes (Figure 4.22 B).

4.2.2.4 Summary of Poly(ADP-ribosyl)ation Metabolism Analyses

In conclusion, these experiments showed that poly(ADP-ribosyl)ation as well as NAD⁺ metabolism are altered in hPARP-1-expressing cells. However, the molecular mechanisms involved seem to be complex and results seem to depend on various factors such as cell-type, intensity of the genotoxic stimulus, and read-out of the experiment.