1 Introduction

The use of ionizing radiation as a therapeutic agent has been recognized for almost a century, and continues to be widely used for the treatment and palliation of many human cancers. Ionizing radiation can also be mutagenic or lethal to individual cells, thus a critical balance must be achieved when using radiation as a form of anticancer treatment to ensure tumor cell death with minimal side effects to normal tissue and organ function. Morphological damage following nonlethal exposure to whole-body ionizing radiation is detectable only in a few select cell types. Histological studies on tissue derived from irradiated mammals reveal immediate and extensive death to specific cells in the spleen, thymus, bone marrow, and intestinal epithelium. This phenomenon of cellular and nuclear disintegration, now defined as apoptosis (1) and long known to be an outcome of whole-body irradiation in these mammalian cell types, has been most carefully studied in spleen, thymus, and intestinal epithelia (2).

In contrast to extensive morphological studies, biochemical pathways that govern the differential survival or repair of normal cells exposed to ionizing radiation in vivo are only beginning to be defined. The discovery that ionizing radiation-induced growth arrest or apoptotic pathway is dependent on the tumor suppressor protein p53 (3), prompted further examination of the response of the p53 pathway to ionizing radiation injury in vivo and provided the most accurate polypeptide marker known whose modification can provide a direct readout of ionizing radiation injury in vivo.

Current methods that detect perturbation of the ionizing radiation-dependent p53 pathway include:

  1. 1.

    Increases in p53 protein levels.

  2. 2.

    Increase in expression of gene products under transcriptional control of p53 protein.

  3. 3.

    Induction of apoptosis in some cell types.

Certain cells of spleen, thymus, bone marrow, and intestinal epithelia are sensitive to ionizing radiation-dependent apoptosis in a p53-dependent manner (4–8). However, most cell types do not suffer any acute morphological defects following radiation injury. Of the organs that do not suffer from radiation-induced death, select cell types activate the transcriptional function of p53, including the lung, kidney (9), and salivary gland duct epithelia (10). In contrast to this variable induction of the p53-dependent apoptotic and transcriptional function in vivo, increases in p53 protein levels after radiation exposure appear to provide a striking readout of radiation injury in vivo. An approximately two- to fourfold increase in p53 protein levels can be observed after radiation damage regardless of organ type, thus providing a relatively unique and sensitive protein marker of radiation injury. This chapter describes sensitive immunochemical and cell-staining methods for quantitating p53 protein in tissues.

2 Materials

2.1 Cell Lysis

  1. 1.

    Lysis buffer: 1% Nonidet P-40 (NP-40), 25 mM HEPES, pH 7.6, 5 mM dithiothreitol (DTT), 50 mM NaF, 0.15 M KCl, 1 mM benzamidine.

  2. 2.

    Liquid nitrogen.

  3. 3.

    Hand-held pestle for 1.5-mL microcentrifuge tubes.

  4. 4.

    Refrigerated (2°C) microcentrifuge.

  5. 5.

    Protein assay kit (Bio-Rad).

2.2 ELISA

  1. 1.

    Antimouse p53 monoclonal antibody (MAb) PAb248 or PAb246 (Oncogene Sciences), rabbit antimouse p53 polyclonal CM5 antibody (Novocastra, Newcastle upon tyne, UK), goat antirabbit horseradish peroxidase (GAR-HRP) IgG antibody.

  2. 2.

    96-Well ELISA plates.

  3. 3.

    0.1 M sodium borate buffer, pH 9.0.

  4. 4.

    PTMB buffer: phosphate-buffered saline (PBS) containing 0.1% Tween-20, 3% milk powder, and 5% bovine serum albumin (BSA).

  5. 5.

    PT buffer: PBS containing 0.1% Tween-20.

  6. 6.

    Capture buffer: 20% glycerol, 25 mM HEPES, pH 7.6, 5 mM DTT, 0.15 M KCl, 0.1% Tween-20, 5% BSA, 3% milk powder, 1 mM benzamidine, and 50 mM NaF.

  7. 7.

    ECL kit (Amersham).

  8. 8.

    Chemiluminescence-ELISA plate reader.

2.3 Gel-Shift Assay

  1. 1.

    DNA binding buffer: 20% glycerol, 25 mM HEPES, pH 7.6, 50 mM KCl, 10 mM MgCl2, 1 mM DTT, 1 mg/mL BSA, 0.1% Triton X-100.

  2. 2.

    T4 Polynucleotide kinase end-labeled oligonucleotides containing the 20-mer p53 consensus DNA binding site (PG): AGACATGCCTAGACATGCCT.

  3. 3.

    Competitor DNA: pBluescript (Stratagene), poly dI/dC, or salmon sperm DNA.

  4. 4.

    Antimouse p53 antibodies PAb421 and PAb248 (Oncogene Sciences).

  5. 5.

    Polyacrylamide gel electrophoresis equipment.

2.4 Immunohistochemical Staining

  1. 1.

    Buffered formalin.

  2. 2.

    Parafin wax.

  3. 3.

    Poly-l-lysine coated slides.

  4. 4.

    Hydrogen peroxide solution: 1% hydrogen peroxide in PBS.

  5. 5.

    10 mM Citrate buffer, pH 6.0.

  6. 6.

    5% Normal swine serum (NSS).

  7. 7.

    Anti-p53 CM5 antibody.

  8. 8.

    Swine antirabbit HRP antibody (DAKO).

  9. 9.

    3,3′ Diaminobenzidine (DAB) solution: Add 1 g of DAB/50 mL of PBS and warm to 50–60°C to dissolve.

  10. 10.

    Developing solution: Add 200 μL of DAB solution to 40 mL of PBS, then add 30 μL of H2O2, and mix. This solution must be prepared immediately before use.

  11. 11

    Hematoxylin (BDH).

  12. 12

    Histoclear (National Diagnostics, Atlanta, GA).

  13. 13

    DPX mounting medium (BDH).

3 Methods

3.1 Cell Lysis

  1. 1.

    Retrieve tissues from nonirradiated or irradiated animals, and freeze immediately in liquid nitrogen. Store at −80°C.

  2. 2.

    Lyse tissues in a frozen state by homogenization in a 1.5-mL microfuge tube using a handheld pestle in approx 3 volumes of lysis buffer. Lysis from frozen cells is important to minimize phosphatase and protease liberation prior to buffer addition. Following a 20-min incubation on ice, sediment the lysate in a refrigerated microcentrifuge (2°C) at 12,000g for 10 min.

  3. 3.

    Recover the soluble supernatant and, prior to storage at −80°C, determine the protein concentration by the method of Bradford (e.g., using a Bio-Rad protein assay kit).

3.2 Nondenaturing ELISA (see Note 1 )

  1. 1.

    Coat individual wells from a 96-well ELISA plate with PAb248 or PAb246 antibody in 50 μL of sodium borate buffer overnight at 2°C. The antibody concentration should be 1 μg/mL or 50 ng of MAb/well (see Note 2 ).

  2. 2.

    Remove the antibody solution by aspiration and block the remaining reactive surfaces by the addition of 200 μL of PTMB buffer.

  3. 3.

    Following incubation at room temperature for 1 h, remove the PTMB buffer and wash the wells four times with PT buffer to remove any remaining unbound MAb.

  4. 4.

    The p53 protein-capture reactions are performed as follows. Prepare an increasing twofold dilution series of the concentrated cell lysate initially starting from 500 μg of protein and continuing to 5 μg of protein in a final volume of 50 μL. Add 50 μL of capture buffer to each dilution, and incubate for 4 h at 0°C.

  5. 5.

    Wash the wells three times with PT buffer to remove the cell lysate, and add a 1∶1000 dilution of CM5 antibody in 50 μL of PTMB buffer. Continue the incubations for 1 h at room temperature.

  6. 6.

    Wash the wells three times with PT buffer to remove unbound primary antibody, and add a 1∶1000 dilution of GAR-HRP secondary antibody in 50 μL of PTMB buffer. Continue the incubations for 1 h at room temperature.

  7. 7.

    Wash the wells three times with PT buffer to remove any unbound secondary antibody.

  8. 8.

    Initiate the chemiluminescence assay by adding 50 μL of ECL solution to each well, and place the microtiter plate within the cavity of an opaque ELISA plate reader. The peroxidase enzyme activity can be quantitated up to 1 h following the addition of the ECL substrate. ECL-based quantitation is essential for the detection of the low levels of p53 protein extracted from tissues.

3.3 Sequence-Specific DNA Binding Gel-Mobility Shift Assay

The biochemical activity of p53 most tightly linked to its biological function involves its ability to bind to a specific DNA sequence and function as a transcription factor. Increases in p53 protein DNA binding activity in response to DNA damage can be quantitated using in vitro sequence-specific DNA binding assays in which p53 protein forms a complex with its 20-bp consensus DNA element. p53 protein activity is difficult to detect in organs owing to its relatively low levels. As such, in vitro DNA binding assays contain the antibodies PAb421 and PAb248 to focus and supershift the p53-DNA complexes to a very large molecular weight that is well separated from contaminating nonspecific proteins that bind to the radiolabeled oligonucleotides in the cell lysate.

  1. 1.

    Set up the following 20 μL reaction for each cell lysate dilution (see Note 3 ):

    a. DNA binding buffer

    10 μL

    b. Radiolabeled PG DNA

    1 ng

    c. Competitor DNA

    Titrate (500 ng to 2 μg)

    d. Anti-p53 antibody

    50 ng

    e. Cell-lysate dilution

    Titrate (50–0.1 μg)

    Incubate at 0°C for 20 min.

    Incubate at 0°C for 20 min.

  2. 2.

    Load the reaction products onto a 4% polyacrylamide gel containing 0.33X TBE and 0.1% Triton X-100, which has undergone pre-electrophoresis at 100 V for 15 min at 3°C. Continue electrophoresis at 200 V for 30–90 min at 3°C.

  3. 3.

    Dry the gel and expose to X-ray film.

3.4 Immunohistochemical Staining of Tissue

In contrast to immunochemistry, immunohistochemistry allows the detection of cell-type-specific induction of p53 in response to DNA damage that is not achieved by other immunochemical methods. This method is of particular use in paraffin sections, since both treated and untreated tissues can be mounted on the same slide to act as an internal control.

  1. 1.

    Retrieve tissues, fix in buffered formalin overnight, and embed in paraffin wax according to standard methods (10). Cut 4-μm sections, and mount on poly-l-lysine-coated slides. Allow to dry overnight at 37°C. Place irradiated and unirradiated tissues side by side on the same slide.

  2. 2.

    Dewax and rehydrate the slides according to the following steps: wash twice in Histoclear for 10 min each. Wash twice in absolute alcohol for 5 min each. Wash twice in methylated spirits for 5 min each. Wash twice in PBS for 5 min each.

  3. 3.

    Incubate the slides in 1% hydrogen peroxide solution on a shaker for 20 min to block endogenous peroxidase activity.

  4. 4.

    Wash the slides twice in PBS for 5 min each.

  5. 5.

    Formalin preservation causes masking of antigens from antibody recognition. To retrieve the antigens, the slides are boiled. Immerse in warm citrate buffer and boil in a microwave at full power (750 W) for 10–20 min. The time required for boiling may vary between microwaves. Therefore, a range of times should be tested to achieve the best results. Do not allow the slides to dry. Cool the slides under slow-running tap water.

  6. 6.

    Wash the slides twice in PBS for 5 min each.

  7. 7.

    Block the slides by covering them with 5% normal swine serum for 30 min in a humidifying incubator.

  8. 8.

    Tap off excess serum. Add CM5 antibody (diluted 1∶5000–1∶10,000) to cover the sections completely. Incubate overnight at 4°C.

  9. 9.

    Wash the slides three times with PBS for 5 min each with shaking.

  10. 10.

    Incubate with swine antirabbit HRP-conjugated antibody (diluted 1∶100) for 1 h in the incubator.

  11. 11.

    Wash the slides as in step 9.

  12. 12.

    Prepare the developing solution, pour it over the slides, and incubate for 5–10 min.

  13. 13.

    Wash the slides three times in tap water, and counterstain with hematoxylin.

  14. 14.

    Dehydrate the slides by passing them back through the alcohol solutions: twice in methylated spirits for 5 min each; twice in absolute alcohol for 5 min each; twice in Histoclear for 5 min each. Mount with DPX resin under a cover slip and view using a light microscope.

An example of this staining showing the difference in p53 levels between irradiated and unirradiated tissues can be found in ref. (10).

4 Notes

  1. 1.

    Alternative procedure: p53 immunoprecipitation protocol. Owing to the low abundance of p53 protein in most organs, a combined immunoprecipitation with MAbs PAb248 and PAb246 and immunoblotting with the polyclonal antibody CM-5 is required to document changes in levels of p53 protein (10).

    1. a.

      Incubate 500 μg of cell lysate by rotating in a mixer for 2 h at 4°C in 200 μL of IP buffer (PBS containing 0.1% Tween-20, 5 mM DTT, 50 mM NaF, 0.15 M KCl, and 1 mM benzamidine) containing 1 μg of PAb248 or PAb246 antibodies covalently crosslinked to 20 μL of protein G beads (Pharmacia). Crosslinking of IgG to protein G beads is required to reduce the background from IgG heavy chain observed when performing immunoblots with polyclonal antibody CM5. Dimethyl pimilimidate is used to cross link the IgG to protein G beads chemically according to previously published methods (11). Briefly, antibody is bound to protein G beads (20 μg of MAb/20 μL of beads), chemically crosslinked, and washed in appropriate buffers.

    2. b.

      Wash the beads three times with 500 μL of IP buffer. Elute the bound protein with SDS sample buffer (4%SDS; 20 mM Tris-HCl, pH 6.8; 10% glycerol; and 0.1 M DTT). Load the solubilized samples onto a 10% SDS-polyacrylamide gel. Following electrophoresis, transfer the protein to a nitrocellulose membrane (Amersham) in transfer buffer (20% methanol; 90 mM glycine; 12 mM Tris-OH) at 20 mA for 18 h or 250 mA for 2 h with cooling.

    3. c.

      Block the protein binding sites on the nitrocellulose membrane by incubation in PTM buffer (PBS containing 0.1% Tween-20 and 5% nonfat milk powder) for 1 h at room temperature on a rotating platform.

    4. d.

      Incubate the blot with CM5 antibody (diluted 1∶1000) in PTM buffer.

    5. e.

      Wash the blot three times in PT buffer, and incubate with GAR-HRP (diluted 1∶1000) or rabbit antimouse antibody linked to HRP for 1 h at room temperature in PTM buffer. Wash the blots three times with PT buffer. Detect peroxidase activity using an ECL kit (Amersham). Approximately 1 s to 5 min are required to detect p53 protein in lysates, depending on the p53 concentration.

  2. 2.

    Although most methods employ the use of MAbs at 30 μg/mL, thus precluding the use of this assay for many laboratories owing to the expense, this amount of antibody is an overestimation of the amount required.

  3. 3.

    When assaying p53 protein in whole-cell lysates, it is important to titrate both the lysate and different types of competitor DNA, and optimize the assay using cocktails of different DNAs. This is owing to the fact that different cell types have different levels of p53 protein, DNA binding proteins, and nucleases that can potentially compete with p53 in binding to the radiolabeled DNA.