Cell Death Analysis (Annexin V/PI)

3x10⁵ HeLa Kyoto and HeLa Kyoto PARP-1 KO1 cells were seeded in 6-well plates and incubated overnight at 37°C. PARP inhibition, if desired, was performed with 10 µM ABT888 30 min before the experiment in incomplete medium. Cells were treated with BPDE in incomplete medium in the presence or absence of the PARP inhibitor (1, 2, 5 and 10 µM BPDE). Camptothecin (50-100 µM) was used as a positive control of cell death induction. After 1 hour the toxicants were removed and the cells were further cultured in growth medium (w/ or w/o ABT888) for 48 h. Then, medium of the samples was taken off and collected in a 50 ml centrifugation tube. Cells were washed with PBS and harvested with trypsin, both of which were subsequently pooled with the medium. The cell suspension was centrifuged with 1,000 rpm for 5 min and the resulting pellet resolved in cold PBS. Using the Casy Cell Counter, the total cell number was determined. 1x10⁶ cells were centrifuged (5 min; 1,000 xg; 4°C), the supernatant discarded and the cells resuspended in 1 ml annexin binding buffer (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2). To 195 µl of cell suspension 5 µl annexin V-FITC was added and incubated in the dark for 15 min. Subsequently, 200 µl PI staining solution (10 mM HEPES/NaOH, pH 7.4; 140 mM NaCl; 2.5 mM CaCl2; 10 µg/ml PI) were added and kept on ice until measurement.

Unstained, as well as PI and annexin V single-stained samples were prepared as well to establish correct gating and fluorescence compensation. Measurement of 20,000 cells was performed with a BD FACSCalibur and the CellQuest Pro 6.0 software and results were analysed with FlowJo 8.8.7.

- 79 - 3.2.5.15 alamarBlue Assay

The alamarBlue assay is a sensitive method based on the cellular reduction of resazurin to resorufin.

This causes a change in the fluorescence spectrum, resorufin is a detectable bright-red fluorescent dye.

The reduction of resazurin takes place in viable cells, using their endogenous redox system by oxidizing NADH to NAD⁺. Thus, the amount of reduced resazurin is an indicator of the cellular redox potential and as a consequence the cellular health. From the amount of fluorescence produced it can be inferred to the number of metabolizing and healthy cells.

After harvesting HeLa Kyoto or HeLa Kyoto PARP-1 KO1/KO2 cells with trypsin, cells were counted thrice and diluted to 6x10⁴ cells/ml. 100 µl/well (6,000 cells) of the cell suspension was distributed to a 96-well plate in technical trip- or quadruplicates and incubated for 3 hours at 37°C to give the cells the chance to adhere. 30 minutes prior to the cell treatment, medium was exchanged to fresh growth medium with or without 10 µM of the PARP inhibitor ABT888. As a positive control cells were incubated in 1 mM H2O2 in PBS/ 1 mM MgCl2 for 5 min at 37°C. For BPDE treatment, 0.01 – 10 µM BPDE were directly before usage diluted in prewarmed, incomplete DMEM. After 1 hour at 37°C medium was exchanged again to fresh growth medium. In case of PARP inhibition, ABT888 was present during and after the BPDE treatment. Cells were incubated for either 24 or 45 hours before 10 % alamarBlue solution was added to each well. After additional 4 hours, in which the healthy portion of the cells reduced resazurin to resorufin, the fluorescence was measured with an Varioskan Flash fluorescence reader (Ex.: 535 nm / Em.: 580 nm).

3.2.5.16 Clonogenic Survival Assay (CS Assay)

HeLa Kyoto and HeLa Kyoto PARP-1 KO1/KO2 cells were trypsinized and harvested as described before. Using a Casy Cell Counter cell numbers were determined by taking the average of three independent countings. Cells were centrifuged (1,000 rpm; 5 min) and resuspended in incomplete medium to a concentration of 2x10⁵ cells/ml. The cell suspension was distributed in 2 ml reaction tubes (1 ml each) and incubated at 37°C with or without 10 µM ABT888. After 10 min 1 µl of the freshly prepared 1,000x BPDE stock solution was added and mixed carefully by pipetting up and down.

Treatment occurred at 37°C for 30 min. Cell suspension was further diluted 1:100 in complete medium before 1,000 cells were seeded in 60 mm petri dishes with or without PARP inhibitor in technical triplicates. Alternatively, ABT888 untreated cells were seeded and incubated for 6 h to allow cell attachment before adding 10 µM ABT888. After 7 days of incubation at 37°C without movement, the cells were fixed with 10 % PFA for 30 min and stained with 0.1 % crystal violet (in PBS) for 45 min.

Excessive crystal violet was removed by repeated washing in DI water and the dishes were air dried and sealed with parafilm. A binocular was used to count colony numbers per dish. Clusters of more than 20 cells were determined as healthy colonies.

3.2.5.17 HPRT Forward-Mutation Assay

Pre-Existing HPRT Mutant Cleansing (HAT Selection):

3x10⁶ CHO cells were seeded in T-160 cell culture flasks and incubated overnight. The next day, medium was removed and replaced by full growth medium, additionally supplemented with HAT (hypoxanthine-aminopterin-thymidine) for HPRT mutant removal. Cells were cultured in this HAT selection medium for 72 h and passaged after 2 days if necessary. On day three, HAT medium was replaced by HT medium (hypoxanthine-aminopterin) and cells were allowed to recover for 24 h.

- 80 - BPDE Treatment and Phenotypic Expression:

Cells were washed with DMEM and PBS, trypsinized as described before, re-seeded in 6-well plates (3x10⁵ cells/well) and incubated overnight. 30 min before treatment, DMEM was changed to medium with or without 10 µM ABT888. CHO cells were incubated at 37°C in incomplete medium with the desired BPDE concentrations (1, 10, 50, 100, 200, 500 nM) in the presence or absence of ABT888.

After 1 h, the BPDE was removed, cells were washed once with PBS and incubated for 23 h in fresh growth medium at 37°C. On the next day, cell numbers were readjusted to 3x10⁵ cells/well and an 11 day phenotype expression period (w/ or w/o ABT888) started, with subculturing every other day.

Toxicity Testing (Clonogenic Survival Assay):

Simultaneously to the phenotypic expression, a clonogenic survival assay was performed to control general BPDE toxicity on CHO cells. For that reason, 450 BPDE treated cells were seeded in 60 mm petri dishes and incubated at 37°C without movement or medium change. When PARP inhibition was desired, 10 µM ABT888 was present during that time. Each sample was prepared as technical duplicate.

After 7 days, the cells were carefully washed once with PBS and fixed with 10 % PFA (in PBS) for 30 min. Colonies were stained 0.05 % crystal violet for another 30 min. Repeated washing steps with DI water followed until all excessive crystal violet was removed. Finally, the plates were air dried, sealed with parafilm and stored in the dark until further analysis.

HPRT Mutant Selection:

After 11 days of phenotypic expression, the mutant selection was started. Therefore, cells were harvested with 150 µl trypsin and transferred with 1.4 ml growth medium to 2 ml reaction tubes. Cell numbers were carefully determined by taking the average of two independent countings. Cells were diluted to 2x10⁴ cells/ml in selection medium (40 µM 6-thioguanine) and seeded in 100 mm petri dishes (2x10⁵ cells) in technical triplicates. Mutant selection went on for 8 days, during which medium and 6-TG was refreshed once on day 4.

Simultaneously, plating efficiency (PE) was analysed by seeding defined numbers of CHO cells in technical triplicates in 60 mm dishes and culturing without selection pressure. As for the mutant selection, cells were as well incubated for 8 days at 37°C, but without a change of medium. 10 µM ABT888 was present at all times in both, mutant selection and plating efficiency medium of samples with desired PARP inhibition.

After 8 days, the cells were carefully washed once with PBS and fixed with 10 % PFA (in PBS) for 30 min. Colonies were stained 0.05 % crystal violet for another 30 min. Repeated washing steps with DI water followed until all excessive crystal violet was removed. Finally, the plates were air dried, sealed with parafilm and stored in the dark until further analysis.

Analysis and Calculation of Mutant Frequency:

Colonies were counted using a binocular, thereby, cell clusters with more than 20 cell in diameter and a close attachment to each other were considered as healthy colonies. Mutant frequency (MF) was calculated as followed:

C = \ Bkik ` j n ^ k − l i B_]o Bk g]_]k l l g g B iil

- 81 -

sb = \ Bkik ` j n ^ l i B_]o Bk g]_]k l C ∗ 2t10^E

For reasons of clearness, mutant frequency was further displayed as mutants per 10⁶ cells:

sb ∗ 10^u = j_\ _l 10v ^u B iil

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4 Results

4.1 Overexpression and Purification of Recombinant Proteins

4.1.1 General Aspects of Recombinant Protein Design

In the course of this thesis the following recombinant proteins were needed for the various biochemical analysis, and thus overexpressed and purified: PARP-1, full-length XPA and five fragments of XPA (F1-F5). For each of these proteins it was possible to draw on to previous work and established purification protocols at varying extent.

For overexpression of PARP-1 and His-XPA, plasmid constructs were on hand and ready for baculovirus recombination. But while for PARP-1 an established gravity flow purification strategy was used, the purification protocol of His-XPA had to be adapted to an ÄKTA FPLC based system. For the fragments of XPA the generation of overexpression constructs and purification strategies had to be established ab-initio (Figure 4.2).

For a detailed biochemical analysis of XPA and the XPA-PAR interaction it was aimed at purifying five XPA-fragments (Figure 4.1). When designing the fragments, it was taken care to keep the multiple functional domains of XPA intact. Three fragments covered the main body of XPA’s aa sequence (F1, F3 and F5), the remaining two overlapped with these, covering their points of contact (F2 and F4). The first fragment represents the N-terminal part of XPA (F1; aa 1-97). It stretches over the CEP164-binding site and further includes the NLS, the RPA2-, GPN1- and ERCC1-binding site. F3 (aa 98-211) spans the central part of XPA, forms its MBD and includes its zinc finger, RPA1- and part of the DDB2-interaction site. The C-terminal part of XPA, the TFIIH-binding domain, is covered by F5 (aa 226-273).

F2 (aa 60-140) overlaps with F1 and F3 and contains the ERCC1-binding site as well as XPA’s zinc finger. F4 (aa 180-247) represents XPA’s PBM, but further includes the interaction site with DDB2 and overlaps with F3 and F5.

Figure 4.1: Scheme of the XPA fragments spanning regions of interest of XPA. Full length XPA is sketched in grey, with some of its key protein domains. The nuclear localisation sequence (NLS) is highlighted in green, the ERCC1-interaction domain in yellow, the minimal DNA-binding domain (MBD) in blue, the PAR-binding domain in brown and the TFIIH-interaction domain in tan. For the design of the XPA fragments it was taken care to completely cover these regions, keeping them intact.

- 83 - 4.1.2 XPA and XPA-Fragments

4.1.2.1 Generation of Expression Constructs of XPA Fragments

Starting point for the generation of expression constructs was a pSL1180::His-XPA plasmid. Fragments were amplified by PCR and by the use of elongated primers, 5’-end BmtI and 3’-end NotI restriction sites were introduced (Figure 4.2).

PCR products as well as the target vector pVL1392::His were digested (NotI/BmtI) and gel-purified to remove the cut-off products. Subsequently, PCR products were ligated into the pVL1392::His vector. The successful integration and the absence of sequence aberrations was ensured by sequencing.

After baculovirus recombination, Sf9 cells were repeatedly inoculated with the respective virus to amplify the titer. Here, it was taken care not to apply baculovirus with a MOI>1 to avoid accumulation of viruses with mutations in their genome. After 3-4 rounds of amplification, the viral titer was tested by serial dilution of the virus and the infection of a defined number of Sf9 cells.

Virus infected cells, expressing the recombinant protein, were identified by immunofluorescence microscopy (Figure 4.3, representative titer determination of XPA-F3). When obtaining a viral titer in a concentration range of ~1x10⁸ pfu/ml, it was used for overexpression of recombinant proteins.

Amplification of the baculovirus construct carrying the XPA-F4 fragment was not achieved. Expression of the fragment seemed to be cytotoxic.

Figure 4.2: Generation of XPA-fragment overexpression constructs. A. Flowchart showing the process of construct cloning from the PCR of the target region up to the ligation into the baculovirus recombination vector pVL1392. B. Agarose gel visualizing the DNA amplification of the five XPA-fragments.

- 84 - 4.1.2.2 Recombinant Protein Test Expression Test Expression of XPA

Test expressions were performed to optimize the expression conditions of recombinant His-XPA in High Five cells. Cells were inoculated in baculovirus (His-XPA) at MOIs of I and III for up to three days. After the indicted times, cells were harvested, lysed and subject to SDS-PAGE and subsequent immunoblotting (Figure 4.4).

Overexpression of His-XPA could be detected 2 days after infection of cells with the virus and expression levels continued to increase until day

three without showing any signs of degradation. Since a MOI of III induced even higher expression levels than an equal virus-cell ratio, it was decided to perform all following expressions at an MOI of III for 3 days.

Test Expression of XPA-Fragments As described for the overexpression of His-XPA, the optimal expression conditions of the XPA-fragments were identified by test expressions in High Five cells at MOIs of I and III for up to 3 days. As for full-length XPA, all three tested XPA-fragments (F1, F2, F3) showed the highest degree of protein expression at day 3 at a virus MOI of III (Figure 4.5, only XPA-F3 is represented).

Figure 4.3: Baculovirus titer determination. Defined numbers of Sf9 cells were seeded in 96-well plates and inoculated with a baculovirus of unknown titer. After three days, cells were fixed and an immunofluorescence analysis of recombinant protein expressing cells was conducted with target protein specific antibodies. Depicted is a representative image of the forth amplification of XPA-F3 baculovirus at a dilution of 1:10. Scale bar represents 50 µm.

Figure 4.4: Test expression of His-XPA in High Five cells. At an MOI of I or III cells were inoculated for up to three days in baculovirus. Cells were lysed and His-XPA expression was detected by immunoblotting.

Figure 4.5: Test expression of His-XPA-F3 in High Five cells. At an MOI of I or III cells were inoculated for up to three days in baculovirus. Cells were lysed and XPA-F3 expression was detected by immunoblotting.

- 85 - 4.1.2.3 Purification of Recombinant Proteins Purification of Full-Length His-XPA

For the purification of His-XPA, an ÄKTA FPLC based two-step purification process was established.

First, it was taken advantage of the poly(His)-tag of XPA using IMAC with HisTrap FF columns. XPA was further purified using HiTrap Heparin HP columns in a second affinity chromatography, in which XPA’s DNA-binding ability was exploited.

His-XPA expressing High Five cells were lysed and genomic DNA was fragmented by sonification.

Cell extracts were precleared by ultracentrifugation and subsequent filtration, diluted in purification buffer containing 5 mM imidazole, and loaded on a HisTrap FF column (Figure 4.6A).

Despite a significant portion of insoluble recombinant XPA (Figure 4.6B, cell debris pellet), sufficient amounts of protein could be found in the cell extract, which very efficiently bound to the column material. Subsequently, the column was rinsed with elution buffer with increasing concentrations of imidazole. XPA was eluted from the column at 200 mM imidazole. The majority of the protein could be found in the fractions A7-12 (Figure 4.6B).

The XPA containing fractions were pooled, diluted in XPA purification buffer C containing 40 mM NaCl, and loaded via ÄKTA FPLC on HiTrap Heparin HP columns (Figure 4.7A). Again, His-XPA was nearly completely trapped on the purification material and no recombinant protein could be observed in the flow through. The column was rinsed with elution buffer containing increasing concentrations of NaCl. XPA eluted at 400 mM NaCl and the majority of the protein could be found in fractions D4-E6 (Figure 4.7B). Purity of the purified protein varied in the different fractions between 50-95 % and were separately processed in the following.

Figure 4.6: His-XPA purification using HisTrap FF columns in combination with an ÄKTA FPLC. After clarification of the crude cell extract, the lysate was subject to a first IMAC purification step with HisTrap FF columns. A.

Chromatogram of the IMAC purification. His-XPA eluted from the column at 200 mM imidazole. B. Coomassie-stained SDS-PAGE (4-20 %) of different preparation steps and elution fractions. Majority of His-XPA (42 kDa) could be found in fractions A7 to A12.

- 86 - Purification of His-XPA Fragments

Three fragments of XPA were purified in the course of this thesis: the N-terminal fragment XPA-F1, the central minimal DNA-binding domain containing XPA-F3 and XPA-F2, covering a region overlapping the C-terminus of F1 and the N-terminus of F3. His-tagged fragments were overexpressed in High Five cells for three days (MOI III), cells were pelleted and flash frozen for storage. Cell pellets Figure 4.7: His-XPA purification using HiTrap Heparin HP columns in combination with an ÄKTA FPLC. After a first HisTrap FF IMAC purification, pooled elution fractions containing His-XPA were subject to a second purification step with heparin sepharose. A. Chromatogram of the affinity purification. His-XPA eluted from the column at 400 mM NaCl. B. Coomassie-stained SDS-PAGE (4-20 %) of input, flow through and elution fractions.

Majority of His-XPA (42 kDa) could be found in fractions D4 to E6.

Figure 4.8: XPA-F1 protein purification. Fragment 1 was purified in two subsequent steps, IMAC and SEC. A.

Chromatogram of IMAC with HisTrap FF (Ni sepharose) columns. Crude cell lysate was cleared and subject to a FPLC-based purification. B. Coomassie-stained SDS-PAGE (4-20 %) of A. Majority of XPA-F1 could be found in elution fractions C11-D10 (17.5 kDa). C. Size exclusion chromatography was used to further purify XPA-F1. The arrow marks the run volume at which F1 eluted from the column. D. Coomassie-stained SDS-PAGE (4-20 %) of C. F1 eluted with a high degree of purity form the SEC.

- 87 -

were lysed, the DNA was sheared by sonification and the cell extract was cleared by ultracentrifugation and subsequent filtration. The filtrate was diluted in XPA purification buffer (5 mM imidazole) and purified via ÄKTA FPLC.

XPA-F1 Purification:

Due to the N-terminal hexahistidine tag, XPA-F1 was first purified by affinity chromatography with Ni sepharose. The cleared cell extract was loaded via an ÄKTA FPLC on HisTrap FF columns and unspecific, weakly bound proteins were removed by washing with elution buffer containing low concentrations of imidazole. Elution of XPA-F1 was achieved by rinsing the column with 500 mM imidazole. The majority of the recombinant protein could be found in fractions C11-D10 (Figure 4.8A and B). Recombinant protein-containing elution fractions were pooled, concentrated, and further purified by size exclusion gel chromatography (SEC). During SEC, proteins are separated according to their molecular mass, large proteins eluting earlier than smaller ones. Identification of the elution peak in SEC chromatogram was difficult (Figure 4.8C, see arrow), but the target protein containing fractions

Figure 4.9: XPA-F2 protein purification. Fragment 2 was purified in two subsequent steps, IMAC and SEC. A.

Chromatogram of IMAC with HisTrap FF (Ni sepharose) columns. Crude cell lysate was cleared and subject to a first FPLC-based purification. B. Coomassie-stained SDS-PAGE (4-20 %) of A. Majority of XPA-F2 could be found in elution fractions B8 and B7 (18.5 kDa). C. Size exclusion chromatography was used for further purification.

The arrow marks the run volume at which F2 eluted from the column. D. Coomassie-stained SDS-PAGE (4-20 %) of C. F2 eluted with a high degree of purity from the SEC column.

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could be determined by Coomassie-stained SDS-PAGE and immunoblotting (Figure 4.8D and Figure 4.16 right panel). While the actual molecular mass of the fragment was calculated to be 12.5 kDa, the apparent molecular weight was 17.5 kDa. Purity of the XPA-F1 containing fractions (E6-F7) extended from 30-95 %. Elution fractions were further processed separately according to their degree of purity.

XPA-F2 Purification:

XPA-F2 was purified as described for F1, with modifications (Figure 4.9). Cell extracts were cleared by centrifugation and filtration, and were loaded via an ÄKTA FPLC on HisTrap FF columns. The recombinant protein bound with a high efficiency to the column, while the majority of the protein impurities could be found in the flow through. Washing the column with 50 mM imidazole already

XPA-F2 was purified as described for F1, with modifications (Figure 4.9). Cell extracts were cleared by centrifugation and filtration, and were loaded via an ÄKTA FPLC on HisTrap FF columns. The recombinant protein bound with a high efficiency to the column, while the majority of the protein impurities could be found in the flow through. Washing the column with 50 mM imidazole already

Im Dokument The Role of Poly(ADP-Ribosyl)ation in the Molecular and Cellular Response to Nucleotide Excision Repair DNA-Lesions (Seite 91-0)