3 Materials and Methods

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3.1 Cell lines

Prostate carcinoma sarcoma DU145 cells were purchased from ATCC (Manassas, USA. This cell line was isolated from a brain tumour of a patient with metastatic prostate cancer by K R. Stone (Stone et al., 1978). The cells carry a mutation for p53 as well as for Bax.

DU145Bax cells were generated as described earlier (Hemmati, et al., 2002 ) in the laboratory using retroviral infection. Briefly, for expression of Bax in the Bax-negative DU145 cells we employed the retroviral vector HyTK-Bax (Weinmann, et al., 1997 ), which contains the human Bax-a cDNA under the control of a CMV promoter. Supernatant of the virus producing packing cell line Fly-18 (Cosset, et al., 1995 ) was used to infect DU 145 cells in the presence of 8mg/ml polybrene (Sigma Chemical Co., Grunwald, Germany). Hygromycin (0.5 mg/ml) was added to the cells for selection 48 h later. After 3 weeks of selection the cells were subcloned. Bax expression of the clones was determined by Western blot analysis as described below.

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DU145Bak cells were generated as described earlier (Radetzki, et al., 2002 ). Briefly, the human Bak cDNA was cloned by PCR amplification from human cDNA using electroporation. Stable transfectants were selected in 1mg/ml G418. Mock transfectants were generated by the use of the empty pcDNA3 plasmid.

DU145 EGFP-Bax and EGFP-Bak cells were generated as described earlier (von Haefen, et al., 2004 ) by transfection using Lipofectamine 2000 (Invitrogen Gibco, Karlsruhe, Germany). cDNA of Bak and Bax were amplified with PCR. PCR products were digested and cloned as BglII/EcoRI fragments into the corresponding sites of the vector pEGFP-C2 (BD Biosciences Clontech, Heidelberg, Germany) to obtain Bax or Bak that carry the EGFP tag at the N-terminus. Stable transfectants were selected by the use of G418 at 1 mg/ml, subcloned and EGFP-Bak or EGFP-Bax expression of the clones was determined by fluorescence microscopy.

Media: DMEM high glucose (4.5g/l) medium supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin and 100µg/ml streptomycin (All from GICO, Karsruhe, Germany).

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HEK293 cell line is a transformed primary human fetal kidney cell line, which constitutively expresses AdE5E1 proteins (Graham et al. 1977). For this reason they were used for adenoviral amplification and titration.

Media: DMEM high glucose (4.5g/l) medium supplemented with 10% FBS, 100U/ml penicillin and 100µg/ml streptomycin.

3.2 Storing stock cells

For constituting stocks of different cell lines, 1x106 cells in 500µl of pure media were supplement with 500µl of a 1:5 DMSO:FBS mix in cryotubes, frozen for 2h at –20°C and then stored at –80°C and subsequently stowed in liquid nitrogen.

3.3 Agarose Electrophoresis and extraction of DNA

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The separation of the DNA bands was performed with 1% agarose gels containing 0.1µg/ml of ethidiumbromid. The agarose gel was run in a BioRad gel chamber with 1xTBE running buffer.

DNA was extracted from the agarose gel with QIAEX II gel extraction kit (Qiagen, Hilden, Germany). The appropriate DNA-band was cut from the gel and was weighted. The gel slice was incubated with 3 times volumes of buffer QX 1 followed by addition of 30µl of QIAEX 2 buffer containing silica beads. The mixture was incubated for 10min at 50°C to allow the DNA to bind to the beads. Next, the solution was centrifuged at 14000rpm for 30 sec and the pellet was washed twice with 500µl of PE buffer. The pellet was air-dried and the DNA was eluted with water.

The concentration of the DNA was determined by photometric measurement at 260nm while water served as a standard.

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10x TBE:

108g Tris

55g boric acid

7.44g EDTA

in 1l aqua dest.

3.4 PCR

DNA was amplified by the polymerase-chain-reaction. The mixture contained 10ng of DNA, 1pmol of each primer, 200µM dNTPs, 10x buffer and 1U Taq polymerase in a total volume of 20µl (all from Invitek, Berlin, Germany).

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To create the DNA fragments the following program was used: 95°C for 10min to activate the polymerase, 25 cycles of, denaturation of the DNA at 95°C for 60s, annealing at 58°C for 60s, elongation at 72°c for 90s.

3.4.1 Primers

E1A sense: 5’ – GAG ACA TAT TAT CTG CCA CGG AGG – 3’

anti-sense: 5’ – TTG GCA TAG AAA CCG GAC CCA AGG – 3’

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(Adesanya, et al., 1996 )

E4sense: 5’ – GTA GAG TCA TAA TCG TGC ATC AGG – 3’

anti-sense: 5’ – TTT ATA TGG TAC CGG GAG GTG GTG – 3’

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(Adesanya, et al., 1996 )

Bimsense: 5’ – GGA TCC ATG GCA AAG CAA CCT TCT – 3’

anti-sense: 5’ – ACC TCT TAC GTA ACT ACG AGA TCT – 3’

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All primers were purchased from BioTez GmbH, Berlin, Germany

3.5 Precipitation of DNA

1:10 volumes of (3.5M) potassium acetate and 2x volume of 100% ethanol was mixed with the DNA and incubated at -20°C for 15min. After that, the mixture was centrifuged at 14000rpm for 30min at 4°C. The pellet was washed 3x with ethanol, dried and finally dissolved in water. 1 OD equals to 50mg/ml of DNA.

3.6 Ligation of DNA

DNA fragments were ligated into plasmids by using T4 DNA ligase. Up to 200ng of DNA were incubated with 50ng of the appropriate vector together with 1U of T4 ligase in the provided 1x ligase buffer over night at 14°C.

3.7 Transformation of bacteria by heat shock

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50µl of heat competent E.coli DH5α bacteria (Clontech, Saint-Germain-en-Laye, France) were incubated with 5µl of DNA on ice for 30min. Then, the bacteria-DNA mix was “heat-shocked” for 90s at 42°C followed by incubation for 5min on ice. 500µl of LB-media was added to the mixture and incubated for 1h at 37°C. Finally, the bacteria were smeared on an agar plate, containing 50µl/ml ampicilin. The plates were incubated over night at 37°C.

LB Medium:

10 g Trypton

5 g yeast extract

5 g NaCl

in 1 l aqua dest.

Agar plates:

15 g Agar

1 l LB-Medium

100 μg/ml ampicillin

3.8 Immunochemistry

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1.5x105 cells were plated on sterile cover slips placed into 6-well plates and infected with adenoviral vectors at 25 MOI after 24h. After three times of washing with PBS, the cells were fixed with ice-cold 1% paraformaldehyde for 30min. The fixed cells were permeabilized with ice-cold 100% methanol for 1min. The cells were again washed with PBS and incubated with blocking solution for 30min at RT. The primary antibodies were diluted in blocking solution, applied on the cells and incubated over night at 4°C, followed by secondary antibodies for 1h at RT. Finally, the slides were washed with PBS and mounted in Dako fluorescent mounting medium (DakoCytomation, Glostrup, Denmark) and dried at 4°C over night.

To stain the mitochondria, the cells were incubated with MitoTracker Green (Molecular Probes, Leiden, the Netherlands) at 37°C for 1h with a final concentration of 500nM.

The nuclei were stained with DAPI (Sigma Aldrich, Munich, Germany) at a final concentration of 0.5µg/ml.

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3.9 Western blot analysis

3.9.1 Preparation of protein samples and determination of the concentration

Cells were harvested by trypsinization and centrifuged at 2000rpm, 4°C for 5min. Pellets were washed with ice cold PBS and lysed in an appropriate amount of lysis buffer on ice for 30min. After centrifugation at 14000rpm, 4°C for 15min the supernatant was collected as protein sample. To separate the cytosolic from the organelle fraction, the pellets were resuspended with a hypotonic lysis buffer and immediately centrifuged at 2000rpm, 4°C for 5min. the supernatant was kept as the organelle fraction and the pellet was incubated in lysis buffer on ice for 30min and treated as described above. To detect cytochrome c release, the cells were separated into a cytosolic and mitochondrial fraction by using “mito buffer”, a specific lysis buffer. Cell pellets were incubated with the “mito buffer” on ice for 5min followed by centrifugation at 2000rpm, 4°C for 5min. the supernatant containing the mitochondrial fraction was kept and subjected to determination of protein concentration as described above. The remaining pellet was incubated with lysis buffer on ice for 30min. the pellets were centrifuged at 14000rpm, 4°C for 15min the supernatant was collected as protein sample representing the cytosolic fraction.

The protein concentration was determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, USA). 10µl of protein was mixed with 200µl of BCA solution (reagent A: reagent B, 50:1) in a 96-well plate and incubated at 37°C for 30min in the dark. The absorption was measured at 620nm. A BSA concentration row served as standard. The protein concentration was calculated based on the standard curve.

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Lysis buffer

2mM EDTA

0.1% SDS

1% Triton-X100

1mM Na3VO4

1mM beta-glycerolphosphate

1 tablet protease inhibitor cocktail (“Complete”, Roche Diagnostics)

Hypotonic buffer:

200mM HEPES pH 7.4

10mM KCl

2mM MgCl2

1 mM EDTA

in 100ml

to 1ml of buffer add prior to use:

7.5µl 100mg/ml digitonin

7.5µl 10mM PMSF

3.9.2 SDS-PAGE

SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a method to separate proteins and peptides according to their molecular weight (Laemmli, 1970 ). The anionic detergent SDS denaturates proteins by wrapping its hydrophobic tail around the polypeptide backbone, giving the protein a negative charge, which is proportional to its length. The reducing agent β-mercaptoethanol in the sample buffer cleaves any disulfide bonds between cysteine residues, resulting in a completely unfolded protein. Like this, the original conformation and charges of the proteins do not have any influence on how they migrate through the gel. The only parameter is their molecular weight. The proteins are concentrated at the end of the stacking gel before entering the separating gel. Electrophoresis was carried out with a Bio-Rad Mini-PROTEAN® 3 system. Gels were prepared with 0.75 mm spacers and 10 or 15 pockets.

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Samples were mixed with 5x sample buffer, boiled for 5min at 95°C and equal amounts of protein (25µg) were loaded on a 14% SDS-PAGE gel. Electrophoresis was performed at 120V for the first 5 minutes and continued with 180V for approximately 1h.

Separating gel

(14%)aqua dest

1.5M Tris/HCl, pH 8.8

actrylamide/bisacrylamide solution (29:1; 40%)

100µl 10%APS

100µl 10%SDS

4µl TEMED

Stacking gel

2.185ml aqua dest.

380µl 0.5M Tris/HCl, pH6.8

375µl actrylamide/bisacrylamide solution (29:1; 40%)

30µl 10%APS

30µl 10%SDS

3µl TEMED

5x sample buffer

260 mM Tris/HCl, pH 6.8

12.5% mercaptoethanol

20% Glycerol

2% SDS

0.01% Bromphenolblue

5x running buffer

100mM Tris

1M glycin

3.9.3 Blotting of the proteins onto nitrocellulose membrane

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Electrophoretically separated proteins were transferred onto 0.2μm nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany) by semi-dry blotting using a Bio-Tad Trans-blot SD transfer cell (Bio-Rad, Munich, Germany). Membranes and filter paper (Schleicher & Schuell GB005, Dassel, Germany) were kept in CAPS buffer for 3min prior to use. 3 layers of filter followed by the membrane, SDS-gel and again 3 layers of filters were placed on the anode and finally covered with the cathode. Blotting was performed at 1mA/cm2 for 1h. Homogenous transfer was verified with ponceau S red staining.

Blotting buffer

10mM CAPS, pH 11

10% (v/v) methanol

Ponceau

0.1% ponceau-S

in 5% acetic acid

3.9.4 Immunodetection

The blotted membranes were blocked for 1h in blocking buffer to avoid unspecific binding of the antibodies and to avoid background staining. Next, the membranes were incubated with primary antibodies diluted in blocking buffer for 1h at RT. After washing three times with blocking buffer, the membranes were incubated for one hour with horseradish peroxidase-conjugated secondary antibodies, diluted in blocking buffer. Finally, the membranes were washed three times with PBST and protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate system (Pierce, Rockford, USA). This system is based on the chemical reaction of luminol with peroxidase and hydrogen peroxide. The light emission emerging of this reaction is captured on film by autoradiography. The membrane was incubated in the ECL solution for 3min (Luminol:peroxide buffer, 1:1) and exposed to a hyperfilm ECL-film (Amersham Biosciences, Buckinghamshire, England) for an appropriate time.

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Blocking buffer

10% casein (Roche, Mannheim, Germany)

0.1% Tween

in PBS

PBST

0.1% Tween

In PBS

0.5% SDS

3.9.5 Antibodies

Primary

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Secondary

3.10 The Adenoviral vector

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The shuttle plasmid pHVAd2-TRE is used to insert the gen of interest into the E1 region of the adenovirus type 5. It consists of 2.6kb and a multicloning site as described in Bett et al., 1994. After modification by Gillissen at al., 2003 this shuttle plasmid additionally contains a CMVmini promoter and a BGH-poly A signal. The tTA expression cassette from pTet-Off (BD Biosciences Clontech, Palo Alto, USA) was first cloned as XhoI-PvuII fragment into pHVAd2 shuttle vector upstream of CMVmini. Bim cDNA fused to an N-terminal myc tag was cloned into pSL1180 (Amersham Pharmacia Biotech, Freiburg, Germany) and then inserted as a BamHI-XbaI fragment into the pHVAd2 vector resulting in TRE-myc-Bim. Finally, the Ad5 vector was fused to TRE-myc-Bim by homologous recombination of pAd-tTA creating pAd5-myc-Bim-tTA.

3.10.1 Homologous recombination

Adenoviral vector was completed by homologous recombination of pHVAd1 containing the adenovirus genome and the shuttle vector pHVAd2 containing tTA in Escherichia coli strain BJ5183 RecBC-sbcB. Transformation of the bacteria was achieved by electroporation, after which the bacteria were grown of agar plates containing ampicilin. Colonies were picked and grown in liquid LB-media also containing ampicilin and incubated at 37°C over night. The adenoviral vector was isolated from the colonies and was verified by digestion with the appropriate restriction enzymes. E.coli DH5α bacteria were transformed with the accurate plasmid for multiplication. Finally, the adenoviral vector was isolated from the bacteria and used for transfection of HEK293 package cells.

3.10.2 Calcium-phosphate transfection

10µg of PacI digested adenoviral DNA were mixed with 25µl of 2.5M CaCl2 and adjusted with water to a total of 250µl. 250µl of transfection buffer were added drop-by-drop to the mix and gently shaken. Upon shaking calcium-phosphate crystals are formed that bind DNA. The total of 500µl was incubated for 30min at RT. Different concentrations were added to HEK293 cells incubated in 1ml serum free medium. After 1.5h 1ml media with 20% FBS was added to the cells. Once plaques were visible in cell layer, the cells were harvested and the crude virus lysate (CVL) was used for virus amplification.

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Transfection buffer:

8g NaCl

370mg KCl

250mg Na2HPO4x2H2O

5g HEPES

in 500ml water

pH 6.75

sterilize

3.10.3 Amplification of the adenovirus

HEK293 cells were plated into 50 150cm2 flasks. 24h hours later the culture media was discarded and 10ml suspension containing CVL and serum free fresh media was added to the cells. After incubation of 1.5h an equal volume of 10ml media with 20% of FBS was added to the cells. Once the cells detached from the flask, showing sign of viral infection, cells and media were collected and centrifuged. 35ml of CVL and the pellet were kept and the rest of the supernatant discarded.

3.10.4 Adenovirus purification

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Before starting the purification, the CVL was thawed and frozen three times. 2.5ml of 1.25g/cm3 CsCl were poured into an Ultra-Clear centrifuge tube (Beckman Instruments GmbH, Munich, Germany) and 2.5ml of 1.5g/cm3 CsCl were slowly added beneath. Then, 7 ml of CVL from the virus purification was added onto this CsCl gradient. Next, the gradient was centrifuged (Beckmann Optima LE 80-K) at 30 000rpm for 2h. After centrifugation 2 bands were visible in the gradient. The virus band (the lower one) was harvested using a 1ml syringe and mixed with 1.35g/cm3 CsCl to a final volume of 3ml. Then, 3ml of 1.35g/cm3 and subsequently 3ml of 1.25g/cm3 CsCl were carefully added creating a gradient. This was followed by over night centrifugation at 30 000rpm resulting in a purified virus band on the gradient. The virus was collected as previously with a 1ml syringe.

A NAP-25 column (Amersham Buchler, Braunschweig, Germany) was washed 4 times with 5ml of adenovirus suspension buffer. 1ml of the virus, gained from the gradient, was applied onto the column. Then, the column was washed with 1.5ml adenovirus suspension buffer. Finally, 2ml of adenosuspension buffer was added to the column and collected into a fresh tube. The purified virus solution was stored as 50µl aliquots at -80°C.

Adenoviral suspension buffer:

135mM NaCl

3mM KCl

1mM MgCl2

100mM Tris/HCl

sterilize

add 10% glycerol

3.10.5 Virus titration

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A dilution series starting with 1µl, which was cut in half in each step, was pipetted into a 24well plate using serum free media for dilution. HEK293 cells were plated into a 24well plate and infected with the pre-prepared dilution series of the virus by using one well per dilution rate in a volume of 500µl. After 1.5h incubation time, 500µl of media containing 20% FBS was added to the cells. Approximately 14 days after infection the plaques of each well were counted. The titer was estimated based on the plaque formation and the dilution rate.

3.10.6 Verification of replication competent adenovirus (E1a-PCR)

For verification of the adenovirus, the E1a region was tested for unwanted recombination. During virus amplification in HEK293 cells the E1a region could be inserted into the adenoviral vector by homologous recombination and enables the virus to replicate. Therefore the adenoviral DNA was amplified for its E1a region by PCR. Detection of the E1a DNA band would make the adenovirus unusable. For purification of viral DNA 1µl of the purified virus was mixed with 20µl of proteinase K-Mix and incubated for 90min at 55°C. 300µl of Phenol/Chloroform/Isoamylalkohol pH 8.0 (ratio 25/24/1) was added to the sample. After vortexing the sample was centrifuged for 5min at 14000rpm. The upper phase was transferred into a new tube and mixed with 300µl of chloroform. This step was followed by centrifugation for 5min at 14 000rpm. The upper phase containing the DNA was transferred into a new tube and the DNA was acquired by precipitation (see 3.4).

The acquired DNA was tested for E1a positivity by PCR and bands were detected by agarose gel electroporation. As a positive control, PCR and gel electroporation were performed for the E4 region, which is part of the adenoviral vector.

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Proteinase K-Mix:

5mM EDTA

20mM Tris/HCl, pH 8.0

0,2%(v/v) SDS

0.25µg/µl proteinase K

3.10.7 Infection of the cells with adenovirus

All media was removed from the cells to be infected and replaced with media without FBS containing the adenovirus at the indicated amounts of MOI. After incubation for 1.5h at 37°C, the same volume of media with 20% FBS was added to the cells.

3.11 Flow cytometry

Measurements with FACS (fluorescence-activated cell sorter) allow detection of single cells according to different parameters, such as size, granularity and density. Cells floating in a sheath liquid are hit by a laser beam (argon ion laser, excitation at 488nm). For each cell passing, it is measured how much light is absorbed (forward scatter, shows the relative size) and reflected (side scatter, represents the relative density and is proportional to the cell granularity). Moreover, different fluorescence emitted by the cells can be quantified in different channels (FL1=537nm, FL2=597nm, FL3=650nm) of the flow cytometer.

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Evaluation of the obtained data was displayed by using histograms. They show the fluorescence intensity versus the cell numbers.

Flow cytometry analysis were performed with FACScan (Becton Dickinson, Heidelberg, Germany) and later evaluated with CellQuest analysis software.

3.11.1 Measurement of apoptosis

One of the typical features of apoptosis is the fragmentation of the DNA, which often includes also the disruption of the nucleus. The cell partially loses its DNA since the cell membrane is getting permeable during apoptosis. So it is known that apoptotic cells have a lower DNA content than normal cells in the G1-phase. Thus, this DNA can be detected as a so-called sub G1 peak and is a marker for apoptotic cells.

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Propidium iodide which intercalates with DNA is used to measure the increased fluorescence of its fluorochrome. The intensity represents the DNA-content of the cell.

After infection with AdBimL or AdBimS 2x105 cells were harvested by trypsinisation at the indicated time points. The cells were fixed on ice in a 2% formaldehyde solution for 30min. After centrifugation for 5min at 1300rpm 50µl ice cold PBS was added to the each sample, followed by 100μl ethanol (-20°C). The samples were kept on ice for 15min. After centrifugation for 5min at 1300rpm the cells were resuspended in PBS containing 40µg /ml DNase-free RNase A (Roche Molecular Biochemicals, Mannheim, Germany) and incubated for 30min at 37°C. Finally, the samples were pelleted and resuspended in 100µl PBS containing 50µg/ml propidium iodide (Sigma, Deisenhofen, Germany) and subjected to analysis in the FL3-channel.

3.11.2 Annexin-V FITC

To distinguish apoptosis from necrosis, cells were stained with Annexin-V-FITC (fluorescin isothiocyanate) and counterstained with propidium iodide (PI). Annexin-V-FITC binds to phosphatidylserine (PS) on the outer side of the plasma membrane. Propidium iodide can only enter cells with a disrupted plasma membrane. Thus, PI and Annexin-V positivity is a sign for late apoptosis or necrosis, whereas cells positive for Annexin-V, but negative for PI are generally defined as early-apoptotic (Vermes et al. 1995, 1997).

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After infection with AdBimL or AdBimS cells were collected at indicated time points. Cells were washed twice with cold PBS and resuspended in Annexin binding buffer at 1 x 106 cells/ml. Next, 2 µl of Annexin-V-FITC (BD PharMingen, Heidelberg, Germany) and 4 µl PI (20 µg/ml, Sigma-Aldrich, Munich, Germany) were added to 100 µl cell suspension. Samples were incubated for 20 minutes in the dark at room temperature. After incubation, 100 µl Annexin binding buffer was added.

Binding buffer:

10mM HEPES/NaOH pH 7.2

140mM NaCl

2.5mM CaCl2

FITC conjugated peptide carry the corresponding sequence of the active site of the caspase and can therefore bind to the active site.

3.11.3 Measurement of the mitochondrial membrane potential

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Intact mitochondria establish a potential in-between the intramembrane space and the matrix created by an electrochemical gradient. Based on this feature of mitochondria kationic and lipophilic dyes like JC-1 (5,5´,6,6´-tetrachloro-1,1´,3,3´-tetraethyl-benzimidazolylcarbocyanin iodide) can accumulate in the matrix. By forming aggregates the fluorescence shifts from green to red. Depolarisation of mitochondria is therefore accompanied by increased green fluorescence and is measured in the FL-2 channel of the FACS.

2x105 cells were incubated with adenoviral vectors at 25 MOI for the indicated time points. Samples were collected by centrifugation and resuspended in 1ml JC-1 solution (2.5µg/ml PBS). After incubation for 30min at 37°C samples were centrifuged at 1300rpm for 5min at 4°C and washed three times with cold PBS. Finally, the cells were resuspended in PBS and immediately analysed.

3.11.4 Caspase activity

Caspase activity was measured for each caspase individually by using their specific cell permeable caspase inhibitors (FLICA, Kit assay, Serotec, Oxford, England). These peptides bind covalently to cysteine residues and thereby block the activity of the individual caspase. Caboxyfluorescein is attached to the peptide contributing the fluorescence. Binding of the peptide to the corresponding caspase leads to an increase of the fluorescence, which is identified in the FL1-channel. As for the measurement, 2x105 cells were harvested after adenoviral infection at indicated time points and resuspended in 500µl PBS with 30µM final concentration of the peptide. Samples were incubated for 20min at 37°C and washed 3 times with washing buffer. Finally, the cells were diluted in 200µl PBS and measured by FACS.

3.11.5 Caspase inhibition

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To inhibit the activity of caspases peptide with a specific sequence are used. They are bound by the active site of the corresponding caspase. For caspase-3 the DEVD-fmk was used, for caspase-9 LEHD-fmk, caspase-8 IETD-fmk (all from Calbiochem, Bad Soden) and for caspase-4 LEVD-fmk (Biovision). To block all the caspases a pan caspase inhibitor zVAD-fmk peptide was used (Calbiochem, Bad Soden).

After infection of 2x105 cells with either AdBimL or AdBimS, they were treated with the corresponding 20µM caspase inhibitor. The cells were collected at indicated time points and prepared for apoptosis measurement as described in 2.6.1

3.11.6 Calcium release

Thapsigargin is a sesquiterpen-lactone which is extracted form umbellifere Thapsia garganica (Ramussen at al., 1978) Thapsigargin is releasing Ca2+ from the ER by specifically blocking SERCA (Ca2+-ATPase of the ER) and none of the other Ca2+-ATPases. Thapsigargin was used as a positive control and subjected to measurement as described.

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Fluo-3/AM (Molecular Probes, Leiden, The Netherlands) is an acetoxymethyl ester, which can pass the plasma membrane if it is uncharged. Inside the cells esterases transform it into a charged molecule. In this state the complex binds Ca2+ ions, accumulates and becomes fluorescence.

Cells were infected with either AdBimL, AdBimS or mock treated and cultured for indicated hours. After harvesting 2x105 cells by trypsinization, pellets were resuspended in 500µl fresh media with 10% FBS. Additionally, 10µl of 2mM Fluo-3AM in DMSO solution was added to each sample. Cells were incubated for 45min at 37°C under vigorous shaking and light protection. After the incubation time, samples were centrifuged for 3min at RT and washed three times with PBS. Samples were immediately analyzed by flow cytometry in the FL-1 channel.

3.11.7 Conformational change of Bax and Bak

2x105 cells were transduced with adenoviral vectors and collected by trypsinization 16h later. After washing with PBS and fixed in 1 ml PBS/0.5% paraformaldehyde (v/v) on ice for 30 min. Again the cells were washed with PBS. N-terminal epitope were detected by conformation specific anti-Bax-NT or anti-Bak-NT antibody (Upstate, Charlottesville, VA, USA). Staining was performed by incubating cells in 1 ml staining buffer (PBS, 1% FCS, 0.1% saponin) containing 0.1 mg of the respective antibody on ice for 30 min. Then, cells were washed in staining buffer, resuspended in 1 ml staining buffer containing 0.1 mg fluorescein-labelled F(ab')2 goat anti-rabbit IgG (H+L) antiserum (Jackson Immuno Research, West Grove, PA, USA) and incubated on ice for 30 min in the dark. After a final washing with PBS the intracellular staining of Bax or Bak was quantified by flow cytometry in the FL1- channel.


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