Investigation of the Proapoptotic Bcl-2 Family Member Bid on the Crossroad of the DNA Damage Response and Apoptosis

Abstract

The BCL-2 family of apoptotic proteins encompasses key regulators proximal to irreversible cell damage. BID, a “BH3-only” proapoptotic family member, plays a critical role in connecting death signals through surface death receptors such as Fas and tumor necrosis factor-α to the core apoptotic pathway at the mitochondria. BID is activated downstream of death receptors by caspase-8 cleavage and N-myristoylation to target mitochondria where it activates BAX, BAK, and the downstream apoptotic pathway. In addition to its role in apoptosis, a role has been uncovered for BID in regulating the DNA damage-induced intra-S phase checkpoint that does not require its death-promoting BH3 domain. Following DNA damage, BID is found in the nucleus where it is phosphorylated by ATM and plays a role in the intra-S phase checkpoint. This checkpoint role is dependent on ATM-mediated phosphorylation at position 78. Thus, BID has two distinct and separable functions: an apoptotic function mediated by caspase cleavage and its BH3 domain and a cell cycle/DNA repair function mediated by phosphorylation by the DNA damage kinase ATM. Studies indicate that the pro-death activity of BID is inhibited by phosphorylation. Taken together, these findings suggest interaction between the two functions of BID. An area of intense research pursuit is determining what dictates how cells respond to DNA damage. Some cells arrest the cell cycle, whereas others undergo apoptosis. We hypothesize that BID acts at the interface between the DNA damage response and apoptosis, in position to signal a cell either to undergo cell cycle arrest and initiate DNA repair or to undergo apoptosis. This chapter describes the techniques used to characterize the role of BID in apoptosis and the DNA damage response.

1. Introduction

The prototype family member BCL2 was identified at the chromosomal breakpoint of the t(14;18) translocation found in 75% of follicular lymphomas and established a third class of oncogenes in which resistance to cell death allows accrual of excess cells, potentially facilitating accumulation of the additional mutations linked with tumor progression (Bakhshi et al., 1985; Cleary and Sklar, 1985; Tsujimoto et al., 1985). Members of the family possess up to four conserved α-helical domains, designated BH1, BH2, BH3, and BH4 (Adams and Cory, 1998; Kelekar and Thompson, 1998). Mutagenesis studies of BCL-2 indicate that the conserved domains are necessary for the interaction with proapoptotic members and for the inhibition of cell death (Yin et al., 1994).

Both pro- and antiapoptotic family members have been identified. The multidomain proapoptotic proteins BAX and BAK serve as a critical gateway in the intrinsic pathway of apoptosis, operating both at mitochondria and endoplasmic reticulum (Wei et al., 2001; Zong et al., 2003). Cells doubly deficient for both BAX and BAK are resistant to multiple death stimuli. The subset of proteins that contain homology only in the death-directing BH3 domain (BH3-only) link upstream death signals to the checkpoint of BCL-2 multidomain members. An emerging paradigm in the field is that these BH3-only proteins may play additional roles, embedded in essential processes within the cell.

Proapoptotic BID is unique among BH3-only BCL2 family members in interconnecting death receptors to the mitochondrial amplification loop of the intrinsic apoptotic pathway. BID was cloned through interaction with BCL2 and BAX (Wang et al., 1996) and was purified biochemically as a protein mediating cytochrome c release from mitochondria following the activation of death receptors (Luo et al., 1998). In vitro studies of mitochondria and recombinant truncated BID indicate that it activates the multidomain BCL2 family members BAX or BAK, resulting in allosteric conformational change and release of cytochrome c (Desagher et al., 1999; Wei et al., 2000).

The role of Bid in normal development and cellular homeostasis has been characterized using mice in which Bid has been disrupted. Bid-deficient mice are viable and execute developmental cell death normally (Yin et al., 1999). When challenged with the agonistic anti-fas antibody, Bid-deficient mice are resistant to the hepatocellular apoptosis that kills wild-type mice, indicating a critical role for BID in this Fas-signaled death. Aging Bid-deficient mice spontaneously develop a myeloproliferative disorder with elevated absolute neutrophil counts, and over time, the mice progress to a fatal clonal disorder resembling chronic myelomonocytic leukemia (Zinkel et al., 2003). Myeloid progenitors from Bid-deficient mice exhibit resistance to death receptor-induced apoptosis and demonstrate a competitive advantage in vivo. These studies indicate an essential role for BID in maintaining myeloid homeostasis and in suppressing leukemogenesis.

In addition to its role in apoptosis, we have uncovered a role for BID in regulating the DNA damage-induced intra-S phase checkpoint that does not require its death-promoting BH3 domain (Kamer et al., 2005; Zinkel et al., 2005)(Fig. 12.1). Following DNA damage, BID is found in the nucleus, where it is phosphorylated by ATM and plays a role in the intra-S phase checkpoint. This checkpoint role is dependent on ATM-mediated phosphorylation at position 78. Thus, BID has two distinct and separable functions: an apoptotic function mediated by caspase cleavage and its BH3 domain and a cell cycle/DNA repair function mediated by phosphorylation by the DNA damage kinase ATM. Studies indicate that the pro-death activity of BID is inhibited by phosphorylation (Desagher et al., 2001). Taken together, these findings suggest interaction between the two functions of BID.

Figure 12.1.

Model for the dual function of BID. Following death receptor stimulation, BID initiates a proapoptotic program at mitochondria. After DNA damage, BID is phosphorylated in the nucleus and plays a role in cell cycle checkpoint control.

Hematopoietic cells are particularly sensitive to low to moderate levels of genotoxic stress relative to other cell types, relying on apoptosis to prevent accumulation of mutations. In order to maintain homeostasis and prevent leukemogenesis following DNA damage, the pathways directing cell cycle checkpoints and apoptosis must be carefully balanced and coordinated. These cells therefore are particularly well suited to the study of the interface between the DNA damage response and apoptosis. Our laboratory has focused our effort on primary hematopoietic cells as well as immortalized myeloid progenitor cells.

To facilitate biochemical studies of the signals governing the interface between the DNA damage response and apoptosis, we use Hox 11 to immortalize myeloid progenitor cells. This technique has the advantage of immortalizing myeloid progenitor cells at a relatively uniform stage of development (Fig. 12.2), with cells predominantly in the promyelocyte to myelocyte stage of development by morphology. In addition, Hox11-immortalized cells display intact DNA damage-induced cell cycle checkpoints, as well as a relatively high percentage of cells in S phase, making them a useful system in which to study the interface between the DNA damage response and apoptosis (Hawley et al., 1994; Zinkel et al., 2005).

Figure 12.2.

(A) Hematopoiesis. All lineages of hematopoietic cells arise from a puripotent stem cell. Progenitor cells then commit to the myeloid or lymphoid lineage (red boxes) prior to terminal differentiation (black box). (B) Cytospin of Hox11-immortalized myeloprogenitor cells. One thousand cells in 50 μl of PBS were centrifuged at 700 rpm for 7 min in a cytospin. Cells were allowed to dry and were then stained with May–Grunwald–Giemsa stain (Sigma).

2. Isolation of Myeloid Precursor Cells from Mouse Bone Marrow Cells

2.1. Materials

Phosphate-buffered saline (PBS), culture medium: RPMI, 10% fetal calf serum (FCS), l-glutamine, and streptomycin/penicillin

Dissection instruments (two sets of forceps/scissors), 70- μm cell strainer (Fisher), 5-ml syringes, 20-gauge needles, 15- and 50-ml conical tubes, tissue culture flasks, tissue culture plates

Cytokines: Stem cell factor (SCF), granulocyte colony-stimulating factor (GCSF), interleukin 3 (IL-3), granulocyte–macrophage colony stimulating factor (GMCSF), Preprotech or R&D

Magnetic beads for lineage depletion [sheep anti-Rat Dynabeads (Dynal Biotech)]

Magnetic stand for Eppendorf tubes (Promega)

Metal stand for Miltenyi magnets (MACS multistand, Miltenyi)

Magnet for MS columns (MiniMACS separation unit, Miltenyi)

MS columns (one column per Sca1+ sample)

2.2. Buffers and media

PBS

RPMI10: RPMI + 10% FCS, l-glutamine, and 100 U/ml streptomycin/penicillin

DMEM20: DMEM + 20% FCS l-glutamine, and 100 U/ml streptomycin/penicillin

Myelocult 5300 (Stem Cell Technologies), l-glutamine, and 100 U/ml streptomycin/penicillin

Infection medium (IMDM): 20% FCS, 100 U/ml penicillin/streptomycin, 2 mM glutamine, 10 ng/ml IL-3, 20 ng/ml SCF, 10 ng/ml GMCSF, and 2 ng/ml GCSF

Red blood cell lysis buffer: 10 mM Tris-HCl, pH 7.2, 0.83% NH₄Cl Staining buffer: 3% FCS in PBS

2.3. Methods

1. Euthanize mice according to the approved method at your institution.

2. To harvest bone marrow, excise the end of the femur using a single-edged razor blade.

3. Flush the bone marrow from femurs of 6- to 12-week-old mice using a 20-gauge needle and 5 ml of RPMI. Control wild-type mice should be strain, age, and sex matched.

4. Centrifuge bone marrow at 1200 rpm for 5 min and remove medium.

5. Lyse red blood cells by resuspending bone marrow cells in 2 ml of red blood cell lysis buffer for 3 min at room temperature.

6. Add 5 ml of RPMI to stop lysis.

7. Centrifuge bone marrow at 1200 rpm for 5 min and remove medium.

8. Proceed to purification of Lin⁻ cells.

3. Isolation of Myeloid Precursor Cells

Myeloid precursor cells (MPCs) are isolated by lineage depletion followed by positive selection of Sca-1⁺ cells by magnetic beads (Miltenyi).

4. Purification of Lin⁻ Cells

1. Resuspend bone marrow cells at a concentration of 10 million per milliliter in staining buffer.

2. Add antibodies to lineage markers [1:100 dilution of biotin-conjugated anti-Gr-1, B220, Ter119; purified anti-CD3 (BD Biosciences)].

3. Incubate cells for 30 min on ice.

4. Wash cells three times in 10 ml of staining buffer.

5. Wash magnetic beads with 10 ml of staining buffer to wash out the azide [sheep antirat Dynabeads (Dynal Biotech)].

6. Add two magnetic beads per cell (concentration of beads is 4 × 10⁸ beads/ ml) to bone marrow cells.

7. Incubate for 30 min on ice.

8. Using a magnetic separation stand (Promega), deplete differentiated bone marrow cells that express lineage markers. Place cells on a magnetic stand in a 1.5-ml Eppendorf tube for 1 min to allow cells bound to magnetic beads to adhere to the side of the tube.

9. Using a plugged Pasteur pipette, remove buffer and nonadherent cells and transfer to another Eppendorf tube.

10. Repeat step 9.

5. Sca-1 Positive Selection

1. Count lineage-depleted cells and resuspend in 0.5% FCS (Sca1 staining buffer) at 10 million cells per milliliter.

2. Incubate with 1/100 volume FITC-conjugated Sca-1 (Pharmingen) for 15 min on ice.

3. Wash three times with 10 ml of staining buffer.

4. Resuspend cells in 0.5% FCS (Sca1 staining buffer) at 100 million cells per milliliter.

5. Incubate cells with 1/10 volume anti-FITC conjugated to magnetic beads (Miltenyi) for 30 min at 4°.

6. Wash cells three times with 10 ml of staining buffer.

7. Resuspend cells in 1 ml staining buffer.

6. Magnetic Separation

1. Place an MS column in a MiniMACS separation unit on the metal stand.

2. Wash with 500 μl of staining buffer.

3. Load cells onto the washed MS column (Milltenyi) by gravity.

4. Reload the flow through.

5. Wash the column three times with 1 ml of staining buffer.

6. Remove the column from the magnet and elute cells with 1 ml of Sca1 staining buffer.

7. Culture of Lineage-Depleted, Sca1⁺ Bone Marrow Cells

1. Centrifuge cells at 1200 rpm for 7 min.

2. Resuspend cells at 10 million cells per milliliter in DMEM20 or myelocult 5300 (Stem Cell Technologies) media supplemented with SCF (100 ng/ml).

3. Add GCSF (100 ng/ml) to the aforementioned culture after 1 day.

4. Grow NIH 3T3 hph-HOX11 retroviral producer cells (Hawley et al., 1994) to 90 to 100% confluence in 100-mm tissue culture dishes. One dish per culture is needed.

5. Irradiate retroviral producer cells with 3000 rad.

6. Change the medium and incubate 24 h before adding myeloid progenitor cells.

7. After 3 days, add myeloid progenitor cells to irradiated NIH 3T3 hph-HOX11 retroviral producer cells in 100-mm tissue culture dishes containing 10 ml of infection medium. Culture for 3 days at 37° in 5% CO₂ (Hawley et al., 1994).

8. Expand the cells growing in suspension in IMDM 20% FCS, 100 U/ml penicillin/streptomycin, and 2 mM glutamine with 10% conditioned medium from WEHI cells as a source of IL-3. The medium should be changed every third day to replenish growth factors and cytokines. These cells should initially be expanded slowly and maintained at a density of 1 million cells per milliliter. Cells have a tendency to terminally differentiate if maintained at a lower density. Cells should be expanded and aliquots of early passages should be frozen for experimental use.

8. WEHI-Conditioned Medium (Source of IL-3) (Warner et al., 1969)

WEHI cells are grown in RPMI/10% FCS:

1. Split cells 1:2 every second day until cells reach a volume of 400 to 500 ml.

2. Remove 10 ml of cell culture in T75 to maintain actively growing cells.

3. Allow large culture of cells to grow for 5 to 7 days until medium is bright yellow.

4. Spin down cells in large centrifuge.

5. Filter supernatant using a 500-ml filter unit.

6. Aliquot into 50-ml conical tubes.

9. Chromosomal Breakage Assays

Bid^−/− leukemias display increased genomic instability, as evidenced by chromosomal translocations and trisomies. To determine whether BID plays a role in maintaining genomic integrity, we evaluated chromosomal integrity following treatment with mitomycin c (Fig. 12.3).

Figure 12.3.

Metaphase spread of mitomycin c-treated Bid^−/− MPCs. Cells were treated with 100 nM mitomycin c for 24 h. The black arrow indicates a quadriradial, the blue arrow indicates a chromosome fragment, and the red arrow indicates a chromosome break.

9.1. Materials

Mitomycin c (Sigma)

Colcemid (Gibco Karyo Max)

0.068 M KCl

Methanol: glacial acetic acid 3:1 (should be made up fresh)

Glass microscope slides (plain glass, not coated with adhesives) (Fisher)

Giemsa stain (Fisher)

9.2. Protocol

1. Treat cells with 100 μM mitomycin c for 24 h.

2. Arrest cells in metaphase with 0.1 μg/ml colcemid.

3. Incubate cells in 0.068 M KCl for 15 min.

4. Add 1/20 volume methanol.

5. Centrifuge cells at 1200 rpm for 7 min.

6. Resuspend cells in 5 ml of methanol:glacial acetic acid (3:1) and incubate for 10 min at room temperature. (The methanol:glacial acetic acid should be made fresh just before use.)

7. Centrifuge cells at 1200 rpm for 7 min.

8. Repeat steps 6 and 7 two additional times. During the third incubation, place cells on ice.

9. Following the last fixation, resuspend cells in 500 μl of methanol:glacial acetic acid (3:1).

10. Drop cells onto glass slides as follows. Drop approximately 30 μl of cells 12 to 15 inches onto a microscope slide (do not use coated slides) held at a 45° angle. We humidify our slides by placing over a 37° water bath for a few minutes prior to dropping.

11. When the drop begins to look grainy, invert slides and hold over a 37° water bath for 2 s (until the slide fogs).

12. Dry slides on a 55° heating block for 5 min.

13. Stain metaphase spreads for 6 to 8 min with Giemsa stain (Gibco).

14. Photograph 50 metaphases per sample and score for chromosomal damage per metaphase as follows: give each chromosome break a score of +1 and each triradial or quadriradial form a score of +2. Then calculate the number of chromosome breaks per metaphase spread.

10. Radioresistant DNA synthesis

10.1. Materials

96-well round-bottomed tissue culture plates

[methyl-¹⁴C]Thymidine (NEN Life Science Products, Inc.)

[methyl-³H]Thymidine (NEN Life Science Products, Inc.)

10% (w/v) trichloroacetic acid

70% ethanol

95% ethanol

25-mm glass microfiber filters (Whatman GF/C)

Scintillation vials (6 ml polyethylene, Perkin Elmer)

Scintillation fluid (Aquasol, Perkin Elmer)

10-place filter manifold (Fisher-FH225V)

¹³⁷Cs irradiator

Scintillation counter

10.2. Methods

1. Label 1 × 10⁵ cells in 200 μl of the appropriate medium with10nCi of [¹⁴C]thymidine for 24h. This prelabeling provides an internal control for cell number by allowing normalization for total DNA content of samples.

2. Remove the medium containing [¹⁴C]thymidine and replace with fresh medium.

3. Incubate the cells for another 24h.

4. Remove medium and replace with fresh medium.

5. Irradiate the cells in a ¹³⁷Cs irradiator. For each cell type, the dose of ionizing radiation should be titrated so that [³H]thymidine incorporation is decreased by 50% following irradiation in the control cells. Generally, cells should be irradiated with between 2 and 10 Gy, although certain cell types may require higher or lower doses.

6. Incubate cells for 1 h.

7. Pulse label cells with 2.5 μCi of [³H]thymidine/ml for 30min.

8. An additional set of control samples should be included that contain only ¹⁴C to allow correction for channel crossover (see Data Analysis, below).

9. Harvest cells, wash twice with PBS, and transfer to Whatman filters that have been placed on the filter manifold.

10. Wash the filters with 5 ml of ice-cold 10% trichloroacetic acid, 5 ml 70% ethanol, and 5 ml 95% ethanol.

11. Air dry the filters and then place in a scintillation tube with 5 ml of Aquasol scintillation fluid.

12. Measure the amount of radioactivity in a liquid scintillation counter. The resulting ratios of ³H counts per minute to ¹⁴C counts per minute, corrected for those counts per minute that were the result of channel crossover, are a measure of DNA synthesis (Fig. 12.4).

Figure 12.4.

Radioresistant DNA synthesis. Cells were labeled with ¹⁴C and treated with a DNA-damaging agent. After 1 h of incubation, cells were incubated with ³H and analyzed in a liquid scintillation counter.

11. Data Analysis

The principle of liquid scintillation counting involves conversion of the kinetic energy emitted by the decay of β particles into ultraviolet (UV) light, producing approximately 10 photons per keV of energy. The intensity of the emitted light is proportional to the initial energy of the β particle. This UV light is detected by the photo cathode photomultiplier tube (PMT) of the liquid scintillation counter and is converted to an electrical pulse that is proportional to the number of photons. The PMT analyzer collects events according to the energy range or channel into which it falls. The energy emission spectra for ³H (mean energy of emission equal to 18.6 keV) and ¹⁴C (mean energy of emission equal to 156) overlap. It is therefore necessary to correct for the events that are the result of channel crossover. This is accomplished as follows.

The scintillation counter should be set to count two channels: channel 1—a low-energy range from 0 to 18.6 keV (³H and some ¹⁴C)—and channel 2—an intermediate-energy range from 18.6 to 156 keV (¹⁴C).

The adjusted ³H counts are equal to (channel 1 counts) – [(channel 2 counts) × (1-¹⁴C fraction)/(¹⁴C fraction)].

The adjusted ¹⁴C counts are equal to the channel 2 counts/¹⁴C fraction.

The ¹⁴C fraction is calculated using the counts from the control sample, containing ¹⁴C only, and is equal to (average channel 1 counts)/(average channel 1 counts + average channel 2 counts).

The rate of DNA synthesis is equal to the ratio of the adjusted ³H to the adjusted ¹⁴C counts.

12. Subcellular Fractionation

The BCL-2 family of proteins represents a key regulatory checkpoint to apoptosis through mitochondria. Proapoptotic BID interconnects death receptor signaling to the core apoptotic machinery at mitochondria through interaction with the multidomain BCL-2 family members BAX and BAK. Following DNA damage, however, BID has been found in the nucleus, is phosphorylated by ATM and/or ATR, and displays increased radioresistant DNA synthesis, suggesting a role in the intra-S phase checkpoint. Subcellular localization thus represents a key mechanism for determining the role of BID in a given cellular environment. We have therefore undertaken subcellular fractionation and immunofluorescence studies to evaluate BID localization within the cell following DNA damage and death receptor signaling.

12.1. Materials

Benzonase nuclease (Novagen)

β-Glycerophosphate (Sigma)

Sodium orthovanadate (Sigma)

Sodium fluoride (Sigma)

Microcystin LR (Sigma)

Complete, EDTA-free protease inhibitor cocktail (Roche)

Hydroxyurea (Sigma)

Triton X-100 (10%)

12.2. Buffers

Buffer A: 10 mM Tris, pH 7.5, 1.5 mM MgCl₂, 10 mM KCl, and 0.5 mM dithiothreitol (DTT)

Buffer B: 20 mM Tris, pH 7.5, 20% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA,

1 mM DTT, and 10% glycerol

Buffer D: 20 mM Tris, pH 7.5, 100 mM KCl, 12.5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, and 10% glycerol

Add 10 mM β-glycerophosphate, 2 mM sodium orthovanadate, 10 mM NaF, 1/50 volume protease inhibitors, and 1 μM microcystin LR to buffers immediately prior to use.

12.3. Methods

1. Treat 30 million cells with DNA-damaging agent of choice. We use 1 mM HU (30 μl of a 1 M stock) for 2 h.

2. Spin down treated and untreated cells in a 50-ml conical tube.

3. Wash once with PBS.

4. Resuspend cells in 200 μl hypotonic buffer (buffer A).

5. Transfer to a chilled Eppendorf tube.

6. Add 1/10 volume of 1% Triton X-100 buffer dropwise with gentle mixing.

7. Spin at 800g for 10 min at 4°.

8. Remove supernatant and put in a chilled Eppendorf tube. This is the cytosolic fraction.

9. Wash the pellet (nuclei) twice in 200 μl buffer A.

10. Resuspend pellet in 100 μl buffer B.

11. Leave on ice for 30 min. Gently mix every 5 min.

12. Centrifuge at 10,000 rpm for 30 min.

13. Centrifuge for 15 min. at 10,000 rpm.

14. Remove the supernatant and place in a chilled Eppendorf tube. This is the soluble nuclear fraction.

15. If the protein is to be immunoprecipitated, dialyze against buffer D.

16. Resuspend the pellet in 100 μl buffer D.

17. Add 0.5 μl benzonase nuclease.

18. Incubate at 4° on a nutator for 30 min.

19. Centrifuge for 15 min at 10,000 rpm.

20. Remove the supernatant and place in a chilled Eppendorf tube. This is the chromatin fraction.

13. Immunofluorescence Staining on Adherent Cells

We generate epitope-tagged BID by introducing the cDNA for BID into the retroviral vectors pOZ-FH-N and pOZ-FH-C. These vectors are a derivative of the MMLV-based pOZ vector contructed by Bruce Howard and colleagues and modified by Nakatani and Ogryzko (2003) to generate BID that is tagged at either the N or the C terminus with FLAG and hemagglutinin (HA). These vectors contain a bicistronic transcriptional unit that allows expression of two proteins from a single transcript to allow tight coupling to the selectable marker, the interleukin-2α chain receptor (CD25). Expression is checked by transient expression in 293T cells followed by SDS polyacrylamide gel electrophoresis (PAGE) and immunoblot for BID protein, as well as FLAG and HA. Retroviral supernatants are generated by transient transfection of the ecotropic packaging cell line BOSC with the appropriate expression plasmid. Tagged BID is introduced into Bid ^−/− mouse embryonic fibroblasts (MEFS) by retroviral transduction, and transduced cells are stained with antihuman CD25 (Caltag) followed by sheep antimouse Dynal beads. Magnetic sorting collects positive cells. Levels of tagged BID protein are verified by Western blot of BID and compared to endogenous BID levels in wild-type MEFS.

We have introduced BID fused to a HA tag into fibroblasts by retroviral transduction. Cells expressing endogenous levels of BID are isolated by flow sorting, and BID expression levels are verified using SDS-PAGE followed by immunoblotting with anti-BID antibodies (R&D).

13.1. Materials

Autoclaved 22 × 22-mm glass coverslips #1 (Fisher)

Polylysine (Fisher)

DMEM10

Six-well tissue culture plates (Sarstedt)

Mitotracker red (Molecular Probes-Invitrogen)

Methanol:acetone, 3:1 (at −20°)

Normal goat serum (NGS; Invitrogen)

PBS (Gibco)

2- μm filters (Millipore)

Poly-l-lysine solution: 50 μg/ml in 10 mM Tris, pH 8.0 [0.02 g poly-l-lysine (Sigma) in 396 ml water and 4 ml 1 M Tris, pH 8.0]

Opti-MEM (Gibco)

Fugene 6 (Roche)

Polybrene 10 mg/ml in sterile water (Sigma)

PE-conjugated anti-human CD25 (Calbiochem)

0.05% trypsin (Gibco)

Vectashield mounting medium (Vector Labs, Inc.)

Alexa-fluor 488-conjugated anti-HA (Molecular Probes)

13.2. Retroviral transduction

1. Plate 293T cells on a 10-cm dish at a density of 1 million cells per plate the evening before transfection.

2. Remove the medium and replace with 5 ml of fresh DMEM.

3. Mix together 250 μl of opti-MEM, 10 μg of plasmid DNA containing FLAGHA-tagged BID, and 60 μl of Fugene 6.

4. Incubate at room temperature for 30 min; a precipitate should form.

5. Add dropwise to the 293T cells and mix gently.

6. Incubate at 37 °C, 5% CO₂, for 48 h.

7. After 24 h, plate Bid^−/− MEFs on a 10-cm dish at a density of 1 million cells per plate.

8. After 48 h, remove the viral supernatant from the 293T cells.

9. Filter viral supernatant through a 0.4- μm filter to remove any cell debris or nonadherent 293T cells.

10. Dilute viral supernatant 1:2 with DMEM, and add polybrene to 4 μg/ml.

11. Remove media from BID^−/− MEFs.

12. Add viral supernatant to MEFs.

13. Remove viral supernatant and replace with fresh DMEM after 24 h.

13.3. Isolation of virally transduced cells

1. After 48 h, remove media and wash once with 5 ml of sterile PBS.

2. Add 5 ml of 0.05% trypsin.

3. Incubate for 5 min at room temperature or until cells begin to lift off of the plate.

4. Add 5 ml of DMEM and pipette cells off of the plate.

5. Spin at 1200 rpm for 7 min.

6. Resuspend in 500 μl of 3% FBS.

7. Add 5 μl of PE-conjugated anti-CD25.

8. Incubate on ice in the dark (cover with foil) for 30 min.

9. Wash three times with 3% FBS.

10. Resuspend in 500 μl 0.5% FBS.

11. Sort for PE+ cells by FACS (we use the Vanderbilt flow sorting facility).

12. Alternatively, cells may be incubated with sheep antirat Dynal beads as used earlier for lineage depletion, and positive cells isolated using a magnet.

13.4. Pretreatment of coverslips for immunofluorescence

1. Work in a tissue culture hood.

2. Place six sterile coverslips into each well of a six-well tissue culture plate.

3. Add 2 ml of polylysine solution

4. Make sure that the entire coverslip is covered with solution; if not, add more polylysine solution.

5. Incubate at room temperature for 30 min.

6. Aspirate off the polylysine solution.

7. Wash three times with PBS.

8. If slides are not to be used immediately, leave the last wash of PBS on the slides and seal the plate with Parafilm.

9. Store at 4°.

13.5. Cell culture

1. Plate 2 to 3 ml of sorted fibroblasts (at a concentration of 1 × 10⁴/ml in DMEM10) into each of six wells of a plate containing coverslips. If the coverslips are not covered completely, add additional medium.

2. Place in incubator for 24 h. Cells should be 50% confluent at the time of staining.

3. Treat cells with death stimulus.

4. Make up media with Mitotracker red.

   Final concentration: 75 nM

   Stock: 1 mM

   Add 0.9 μl per 12 ml medium

5. Remove medium from cells and replace with medium containing Mitotracker red.

6. Incubate 30 min.

7. Remove medium with Mitotracker red.

8. Wash three times with PBS.

9. Add 4 ml of methanol:acetone (3:1) and incubate at −20° for 10 min.

10. Wash three times with PBS.

13.6. Blocking

1. Make up blocking solution: 5% NGS in PBS.

2. Spin at 13,000 rpm for 2 min to remove particulate matter.

3. Block for 1 h at room temperature. Make certain that the slides remain covered with blocking solution for the entire incubation period.

4. Wash three times with PBS.

13.7. Primary antibody

1. Dilute the primary antibody in 5% NGS (anti-HA Alexa Fluor 488, 1:100)

2. Spin at 13,000 rpm for 2 min.

3. Apply to slides using a p200. We use 200 μl of antibody to cover a 22-mm² slide.

4. Incubate at room temperature for 1 h.

5. Remove the primary antibody. We save the primary antibody and reuse it one time.

6. Wash three times with PBS by rocking the plate gently for 10 min.

7. Blot the edges of the slide with a Kimwipe; do not let the slide dry completely.

8. Pipette 10 μl of Vectashield (Vector Labs) onto a microscope slide.

9. Invert stained coverslip onto the microscope slide.

10. Seal the coverslip using clear nail polish.

11. Staining may be visualized using a fluorescent microscope. We use a Nikon Eclipse E600 microscope.

12. Slides may be kept at 4° for up to 1 week.

14. Annexin V Staining

BID performs two distinct roles, an apoptotic role directed by caspase cleavage and interaction with other Bcl-2 family members through the BH3 domain and a cell cycle checkpoint/DNA repair role directed by phosphorylation by ATM and/or ATR. To study the apoptotic response of cells harboring wild-type BID or BID mutated in one of the aforementioned domains in response to DNA-damaging agents, we have used annexin V/PI staining. During the early stages of apoptosis, cells expose phosphatidylserine on the surface of their plasma membrane. Annexin V is a phospholipid-binding protein that, when conjugated to a fluorophore, has been used to detect exposed phosphatidylserine by flow cytometry. Propidium iodide (PI) is a dye that is excluded from living cells, but is incorporated into DNA when the cell membrane becomes permeable late in apoptosis (Fig. 12.5).

Figure 12.5.

Flow cytometry analysis of annexin V/PI staining. One million MPCs were treated with etoposide for 24 h. Cells were stained with annexin V FITC and PI, and flow cytometry was performed on a BD Facscalibur flow cytometer.

14.1. Materials

3% FBS in PBS

Propidium iodide (Sigma) stock solution is 50 μg/ml in PBS

Annexin V FITC (Biovision, Inc.)

FACS tubes (BD Falcon

10 × staining buffer: 0.1 M M HEPES, pH 7.4; 1.4 M NaCl; 25 mM CaCl₂. Dilute to 1× prior to use.

14.2. Methods

1. Spin down 1 million cells.

2. Resuspend in 100 μl staining buffer + 0.5 μl annexin V FITC.

3. Incubate 30 minutes at room temperature in the dark (cover with foil).

4. Put into FACS tube.

5. Add 100 μl 3% FBS.

6. Immediately prior to analysis, add 4 μl of 50 μg/ml PI.

14.3. Controls

1. No annexin V

2. No PI

15. Conclusion

The assays described in this chapter have allowed us to define a dual role for BID in regulating cell death following death receptor stimulation and a cell cycle checkpoint/ DNA repair role following DNA damage. An area of intense research interest is determining what dictates how cells respond to DNA damage. Some cells arrest the cell cycle and initiate DNA repair, whereas others undergo apoptosis. BID, with its dual roles in the interface between these two pathways, is well positioned to play a key role in determining the fate of a cell following DNA damage.