ARTICLE |
Correspondence to: Albert A. J. Verhofstad, Dept. of Pathology, University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen, The Netherlands.
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Summary |
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Ornithine decarboxylase (ODC), a regulatory enzyme of polyamine biosynthesis, is involved in cell growth and differentiation. Lack of information about the exact cellular and subcellular localization of ODC is one of the main obstacles to precise interpretation of the biological roles of the ODC/polyamine system. Here we describe the development and optimization of an immunocytochemical method to detect ODC in cells and tissues. For this purpose a monoclonal antibody (MP16-2) against a defined epitope of ODC protein was developed. Specificity of the antibody for ODC was substantiated by Western blotting and ELISA analysis using cell and tissue homogenates. In cultured cells, optimal staining results were obtained after fixation with crosslinking fixatives followed by permeabilization with methanol. In rat tissues, ODC immunoreactivity was best preserved in paraffin sections fixed with Bouin's fixative. Antigen retrieval using SDS and citrate buffer substantially increased ODC immunostaining and decreased background staining. Localization studies of ODC in different cell lines showed that strongest staining for ODC was found in the nucleoplasm of mitotic cells, whereas confluent cells showed moderate perinuclear staining. Immunocytochemical studies of various rat tissues showed high cytoplasmic immunostaining of ODC in epithelial cells of kidney, prostate, and adrenal medulla of testosterone-treated rats, in glandular epithelium of small intestine, and in pancreas of neonatal and adult rats. (J Histochem Cytochem 47:13951404, 1999)
Key Words: ornithine decarboxylase, monoclonal antibody, immunocytochemistry, tissue fixation, cell lines, rat
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Introduction |
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Ornithine decarboxylase (ODC, EC 4.1.1.17) is the initial and regulatory enzyme of polyamine synthesis, converting the amino acid ornithine into the polyamine precursor putrescine. Polyamines are organic bases that play an essential role in cell growth and differentiation (
Despite extensive biochemical and molecular biological research, the precise role of the ODC/polyamine system in cell physiology remains to be clarified. Insight into the exact cellular and subcellular localization of ODC might provide valuable information on the role of this metabolically important enzyme. Efforts to assess the (sub)cellular localization of ODC by immunocytochemical means have been made in the past (our unpublished work). However, different localization patterns were found, depending on the cell system studied and the antibody used. These patterns varied from a cytoplasmic or (peri)nuclear pattern to a both cytoplasmic and nuclear localization. An explanation for these discrepancies may be the lack of specificity of the ODC antibodies that were used. Furthermore, effects of cell and tissue preparation (e.g., fixation procedures, permeabilization, dehydration, and embedding in paraffin) on the preservation and/or accessibility of the ODC protein for the antibodies were hardly defined.
To develop a reliable method for immunocytochemical detection of ODC, we raised a specific and potent mouse monoclonal antibody (MP16-2) to a synthetic hexadecapeptide (P16) corresponding to a computer-calculated epitope of ODC (amino acid residues 345360) (
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Materials and Methods |
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Antibody
A mouse monoclonal antibody (MAb) against a defined epitope of ODC was produced and characterized as described previously (
Cell and Tissue Preparations
Human amnion WISH cells (American Type Culture Collection; Rockville, MD), HCT 116 human colon cancer cells (American Type Culture Collection), and NIH 3T3 mouse fibroblast cells (American Type Culture Collection) were cultivated in Dulbecco's modified Eagle's medium (DMEM; Flow Laboratories, Zwanenburg, The Netherlands) supplemented with 10% fetal calf serum (FCS; Integro BV, Zaandam, The Netherlands), 100 IU/ml penicillin, and 100 µg/ml streptomycin (ICN Biomedicals; Costa Mesa, CA). A solution of 0.5 mg/ml trypsin (Difco Laboratories; Detroit, MI) and 0.2 mg/ml ethylene di-amino tetra-acetic acid (EDTA) in PBS was used to detach cells for passage.
To investigate ODC immunoreactivity in ODC-overexpressing cells, NIH 3T3 cells were transiently transfected with an expression construct (12.3 kb) carrying the full-length gene encoding human ODC driven by gut-specific promoter sequences. Transfected cells showed elevated levels of ODC mRNA (two- to 20-fold) and enzyme activity (3.5- to 40-fold) compared with nontransfected cells (manuscript in preparation).
Cell homogenates were prepared for immunochemical analysis (Western blotting, ELISA) by homogenization of 1 x 105 cells in lysis buffer (50 mM Tris, 1 mM EDTA, 0.3% SDS, pH 7.2). After clarification of the lysates by centrifugation at 12,000 x g at 4C for 10 min, the supernatant was used for further analyses.
We purified ODC from kidneys of testosterone-treated NMRI mice as described by
Western Blotting
Homogenates were boiled with sample buffer [62 mM Tris-HCl, pH 6.8, 10% glycerol, 2% sodium dodecyl sulfate (SDS), 650 mM ß-mercaptoethanol, 0.025% bromophenol blue] for 5 min at 100C. The samples and molecular weight markers (Pharmacia) were loaded on 10% SDS-polyacrylamide gels (acryl/bisacryl 29.2/0.8) and subsequently electrotransferred onto nitrocellulose sheets (Millipore; Bedford, MA) in blotting buffer (25 mM Tris-HCl, pH 8.6, 192 mM glycine, 20% methanol, 0.02% SDS).
Blots were washed with PBS containing 0.05% Tween-20 (Merck; Darmstadt, Germany) (PBST) and incubated for 3 hr at room temperature (RT) in PBST containing 5% low-fat milk powder to block nonspecific binding sites. The blots were rinsed in PBST and incubated overnight at RT with MP16-2 antibody (1 µg/ml) in PBST. After washing three times for 10 min in PBST, blots were incubated with secondary antibody, i.e., peroxidase-coupled goat anti-mouse IgG (Dako; Glostrup, Denmark) diluted 1:2000 in PBST. Finally, bound antibodies were visualized using a chemiluminescent detection system (Boehringer Mannheim; Almere, The Netherlands) followed by exposure to radiographic film (Kodak X-OMAT-R) for 1030 sec.
ELISA
ELISA analysis was performed as described previously (M-PO; Dako) diluted 1:1000 in PBST was performed for 1 hr at 37C. After rinsing, wells were filled with a solution of the chromogen 5-aminosalicylic acid (1 mg/ml) (Sigma) and H2O2 (0.25%) in 50 mM phosphate buffer (pH 6.0) and incubated for 30 min at RT. Production of the final reaction product was measured by reading the absorbance at 450 nm in an ELISA plate reader (Titertek; Flow Laboratories).
Artificial Model Preparations
To investigate the effects of the immunocytochemical procedure on P16 antigenicity, the so-called defined antigen substrate spheres (DASS) system was used (
First, the free amino groups of the Sepharose beads were activated with a stoichiometric amount of 6-(1-maleinimido)hexanoic acid 1-succinimidyl ester (MHS; Fluka, Buchs, Switzerland) in 1 ml N,N-dimethylformamide (DMF; Merck) for 30 min. Beads were then rinsed with DMF, treated with acetic acid N-hydroxysuccinimidic ester (AcONSu; Merck) in DMF for 10 min to block nonactivated amino groups and again rinsed with DMF. For the coupling of 1 ml activated beads 5 mg decapped P16 was used. P16 was dissolved in 100 µl DMF/methanol, 14:5 (v/v) and added to the activated beads. After 1 hr, beads were rinsed with PBS and stored at 4C. Intact bead preparations were used for immunocytochemical model experiments. Alternatively, beads were embedded in paraffin and 4-µm-thick sections from the paraffin blocks were mounted on object slides.
Fixation Methods
Crosslinking and coagulant fixatives were tested with the use of artificial model preparations and intact cells. Crosslinking fixatives were phosphate-buffered solutions of 2 and 4% paraformaldehyde (pH 7.3), 4% paraformaldehyde with 0.1% glutaraldehyde, Zamboni's fixative, and Bouin's fixative. Fixation was performed for 1 hr at 4C. The coagulating fixatives were methanol, ethanol, and acetone with an incubation time of 30 min at -20C.
Immunocytochemistry
Artificial Models and Cultured Cells.
Artificial model preparations were made of P16-coupled Sepharose beads that were air-dried on object slides. For intact cell preparations, cells were grown on 8-well chamber slides until 80% confluency. After washing with PBS, slides were incubated with the appropriate fixative. After washing, cells were permeabilized with methanol, ethanol, or acetone series (100, 50, 25%) each step 15 min at -20C. To decrease aspecific binding of antibodies slides were acetylated using a triethanolamine solution (0.1 M triethanolamine, pH 8.8, acetic acid anhydride 0.25%) (MPO; Dako) or rat anti-mouse immunoglobulins conjugated with fluorescein di-isothiocyanate (R
MFITC; Dako). Primary as well as secondary antibodies were diluted in PBS containing 2% BSA and 0.1% Triton X-100. Specificity was investigated by preabsorbing MP16-2 antibody solution with various concentrations (0, 0.1, 0.5, 1, 10 µg/ml) of P16-coupled thyroglobulin for 30 min at RT. Immunoglobulin type-matched mouse MAbs against S-100 (M
-S100; IgG2a) and follicle-stimulating hormone (M
-FSH;IgG1) (Biogenex Laboratories; San Ramon, CA) or dilution buffer were used as negative controls. Slides were incubated with primary (diluted 1:100) or secondary antibodies (diluted 1:300) for 90 min at RT and subsequently washed with PBS. Peroxidase was visualized using 0.06% diaminobenzidine and 0.015% H2O2 in PBS. Object slides incubated with R
MFITC were mounted in a mixture of glycerol (Merck) 90%, Tris-HCl (Merck) 10%, NaN3 (Merck) 0.1%. All other slides were mounted in Permount (Fisher; Fair Lawn, NJ). Slides mounted in Permount were stored at RT. Slides used for immunofluorescence were stored in the dark at 4C.
Staining results were examined using a Zeiss Axiophot microscope (Carl Zeiss; Oberkochen, Germany). Confocal laser scanning microscopy was carried out using a Nikon Diaphot (Nikon; Tokyo, Japan) and a Bio-Rad MRC 1000 (Bio-Rad; Hemel Hampstead, UK) equipped with a kryptonargon laser as described previously (
Tissues.
Adult male rats (Wistar) were treated with testosterone by SC implantation of a timed-release pellet containing 50 mg testosterone (Innovative Research of America; Sarasota, FL). After 72 hr the rats were sacrificed by cervical dislocation and adrenals, colon, kidney, liver, pancreas, prostate, small intestine, and spleen were removed. In a separate experiment, pancreas and intestinal tissues of 1-, 7-, 14-, 21-, 28-, and 100-day-old rats were obtained. Specimens of the tissues were fixed overnight at 4C in 4% paraformaldehyde or Bouin's fixative and paraffin-embedded. Sections 4 µm thick were mounted on object slides, dewaxed, rehydrated through a series of ethanol solutions, and treated with 3% H2O2 in methanol for 3 min to block endogenous peroxidase activity. For heat-induced antigen retrieval (
Slides were incubated with MP16-2 antibody (1 mg/ml) diluted 1:100 in antibody buffer (PBS containing 1% BSA, 0.1% Tween-20, and 0.01% SDS) for 1824 hr at 4C. Specificity of the staining method was investigated by preabsorbing MP16-2 antibodies with various concentrations (0, 0.1, 0.5, 1, 10 µg/ml) of P16-coupled thyroglobulin for 30 min at RT. Bound antibodies were visualized with ABC Elite Vectastain reagents (Vector Laboratories; Burlingame, CA) using H2O2 and diaminobenzidine as chromogen. Sections were placed in a developing medium containing 0.006% H2O2 and 1 mg/ml diaminobenzidine and incubated for 10 min at RT.
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Results |
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Characterization of the Antibody
Western blotting analysis was used to evaluate the immunoreactivity of MAb MP16-2 towards ODC in different cell and tissue preparations. The antibody reacted with a 53-kD protein of the same molecular weight (Mr) as ODC (Figure 1, Lanes 13). ODC-transfected NIH 3T3 cells expressed high levels of ODC protein (Figure 1, Lane 1) compared to nontransfected cells, which showed only a very weak band (not shown). In mouse kidney cytosol, other weaker bands with higher Mr are present, which may represent complexed forms of ODC (Figure 1, Lane 2). The antibody did not react with purified ODC complexed with [3H]-DFMO (Figure 1, Lane 4), indicating that binding of DFMO masks or changes the epitope recognized by MP16-2. ELISA studies showed that the MAb MP16-2 detected ODC in cell and tissue homogenates adhered to plastic surfaces (Figure 2B and Figure 2C). One microgram of antibody was able to detect 60 µg of cell homogenate and 6.5 ng of ODC purified from stimulated mouse kidney, which corresponded to 3.5 pU and 330 pU of ODC activity, respectively. For comparison, Figure 2A shows the ELISA standard titration curve for P16 peptide conjugate of thyroglobulin.
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Methodological Studies
The effects of different fixatives on MP16-2 immunostaining in artificial model preparations (DASS system) and the WISH cells are shown in Table 1. All crosslinking fixatives used were capable of retaining antigenicity of P16 in the DASS system (Figure 3), whereas the use of coagulating fixatives did not result in any immunostaining with MP16-2. Comparable results were obtained in experiments with cultured WISH cells. Fixation with 4% paraformaldehyde or Bouin's fixative provided the best results in intact cells.
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The effects of fixation time, permeabilization procedures, and addition of glutaraldehyde in various concentrations to the 4% paraformaldehyde-containing fixative on immunostaining of P16-coupled Sepharose beads or WISH cells are shown in Table 2. The effects on intact beads or paraffin sections of beads were similar. In cultured cells, optimal staining results were achieved after 1 hr of fixation. After prolonged fixation times, staining intensity decreased. Treatment with a methanol series proved to be the best procedure to permeabilize cells, whereas all permeabilization methods tested did not affect immunostaining of the artificial model preparations. Glutaraldehyde concentrations higher than 0.1% decreased staining intensity in artificial model preparations as well as cultured cells (not shown).
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Beads were not stained after incubation with control antibodies instead of the MP16-2 antibody (Figure 3, inset), whereas cultured cells showed nonspecific background staining. However, treatment of the cells with an acetylating solution as described by
Because fixation with 4% paraformaldehyde or Bouin's fixative provided the best results in the model studies, these fixatives were applied to tissue sections. ODC immunoreactivity was best preserved in rat tissues with fixation according to Bouin. Addition of 0.01% SDS to the antibody buffer solution increased the staining intensity considerably. Further increase in staining intensity and reduction of background staining were achieved with the antigen retrieval procedure using citrate buffer.
Localization Patterns
Typical immunocytochemical staining patterns are shown in Figure 3 and Figure 4. P16-coupled beads were positively stained after incubation with MP16-2 antibodies (Figure 3). Control incubations were negative (Figure 3, inset). Staining was restricted to the periphery of the beads and the intensity was heterogeneous among beads.
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MP16-2 immunostaining studies of different cell lines showed that all cells were positive for ODC. Immunostaining was substantially increased in ODC-transfected cells (Figure 4A) compared with nontransfected cells (Figure 4B). Mitotic cells were more intensely stained than confluent cells (Figure 4C4F). In confluent cells, ODC staining was predominantly cytoplasmic, particularly in the perinuclear region, as revealed by confocal scanning microscopy (Figure 4F).
Distribution patterns of ODC immunoreactivity in rat tissues are summarized in Table 3 and Figure 5. In testosterone-treated rats, strong cytoplasmic staining was found in proximal tubules of kidney (Figure 5C), acinar cells of prostate, and adrenal medullary cells. In the small intestine of 7-, 14-, and 21-day-old rats, immunoreactivity was present in both crypts and villi, whereas in 28-day-old and adult (100-day-old) rats (Figure 5A) strong positivity for ODC was found in crypts only. In the pancreas of neonatal (1-day-old) rats, islets of Langerhans showed strong cytoplasmic staining (Figure 5B). In the pancreas of 14-day-old rats, heterogeneous staining of exocrine cells was observed (
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Preabsorption with 1 µg/ml of P16 peptide or more completely eliminated staining of cultured cells and all tissues investigated (Figure 4C, Figure 4D, and Figure 4D inset). No distinct differences were observed between results obtained with immunoperoxidase and immunofluorescence visualization methods.
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Discussion |
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The purpose of this study was to systematically develop and optimize a reliable method for in situ detection of ODC utilizing an MAb (MP16-2) directed against an artificial antigen representing an ODC epitope (P16; amino acid residues 345360). Authenticity of the P16 ODC epitope was proved by comparing the selected sequence with the primary sequences of other proteins. The sequence -WGPTC-, recognized by MP16-2, is present in all known eukaryote ODCs (
Our ELISA experiments showed that the MP16-2 antibody could also be used to detect ODC in homogenates of cells or tissues coated to plastic surfaces. The difference in calculated sensitivity between the different ODC preparations (purified ODC, cell homogenate) might be due to variations in the amount of ODC epitope that became attached to the plastic surfaces.
In contrast to immunochemical procedures, e.g., ELISA, immunocytochemical staining procedures take place in a far more chemically complex matrix. Model systems using artificial matrices have proved to be valuable tools to study the complexity of (immuno)cytochemical processes (
In contrast, if coagulating fixatives were used the intensity of immunostaining diminished. A frequently occurring problem with coagulative fixatives is that antigens are precipitated and extracted from biological matrices (
Intact cells have to be made permeable to antibodies, especially after fixation with crosslinking fixatives that are known to create diffusion barriers (
The preservation of the epitope by fixatives containing both 4% formaldehyde and 0.05% glutaraldehyde opens perspectives for the application of MP16-2 in electron microscopic studies.
On the basis of the results obtained in the model studies, we tested the MP16-2 antibody on rat tissues fixed with 4% paraformaldehyde or Bouin's fixative. Initial experiments resulted in rather low and poorly reproducible immunostaining of the tissues. With the addition of SDS to the antibody-containing solution, staining was dramatically increased. This observation is compatible with reports from the literature showing that immunogenicity of a number of antigens is retrieved after SDS treatment of cell and tissues (
These observations illustrate the increasing complexity of biological matrices in tissues compared to single cells and artificial models. Antigens in tissues, although well preserved after fixation, might be masked or linked to other components (e.g., by hydrogen or disulfide bonds) and therefore hardly accessible to antibodies.
Although the present study was not primarily meant to investigate the intracellular localization of ODC in cultured cells, interesting results were obtained. Under optimal conditions, ODC showed a similar localization pattern in all tested cell lines. In resting cells, ODC staining was moderate and predominantly in the perinuclear region, whereas mitotic cells were strongly and homogeneously stained. Previous studies on stimulated murine macrophage-like RAW 264 cells (
The optimized immunocytochemical method was also applied to various rat tissues. In testosterone-treated rats, intense staining of ODC was detected in proximal tubules of kidney and acinar cells of prostate, which is in agreement with previous biochemical, enzyme cytochemical, and hybridocytochemical studies (
ODC expression has been shown to change during growth and maturation of developing tissues. In developing rat pancreas and small intestine, we observed age-related localization patterns that might reflect the changes of ODC expression during growth and maturation of these organs (manuscript in preparation).
In conclusion, this report describes conditions required for an optimal detection of ODC with the MP16-2 monoclonal antibody in homogenates, intact cells, and tissue sections. Using optimized techniques, we demonstrated high ODC immunoreactivity in cells and tissues that are engaged in growth or differentiation processes. Moreover, the ELISA technique provides a new and valuable quantitative assay for ODC protein in homogenates of cells and tissues.
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Acknowledgments |
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Supported by the Dutch Cancer Society (grant NKB-93-599) and the Nijbakker Morra Foundation.
We gratefully acknowledge the help of Dr Martin Sauerbeck and Prof Dr Peter Bohley (Department of Physiological Chemistry, University of Tübingen, Germany) in the preparation of purified ODC. We thank Rob Rutten, Xander de Haan, Birgitte Walgreen, and Wilma Van Staveren for technical assistance. We are also grateful to Prof Dr Ron Van Noorden and Dr Wilma Frederiks (Department of Cell Biology and Histology, University of Amsterdam) for valuable comments and suggestions for this manuscript.
Received for publication March 12, 1999; accepted June 1, 1999.
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