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Immunocytological and Preliminary Immunohistochemical Studies of Prothymosin , a Human Cancer–associated Polypeptide, With a Well-characterized Polyclonal Antibody

Persefoni Klimentzou, Angeliki Drougou, Birgit Fehrenbacher, Martin Schaller, Wolfgang Voelter, Calypso Barbatis, Maria Paravatou-Petsotas and Evangelia Livaniou

Institute of Radioisotopes and Radiodiagnostic Products, National Centre for Scientific Research "Demokritos," Aghia Paraskevi, Athens, Greece (PK,MP-P,EL); Pathology Department, Hellenic Red Cross Hospital, Athens, Greece (AD,CB); Universitaets-Hautklinik, Tuebingen, Germany (BF,MS); and Interfakultaeres Institut fuer Biochemie, Eberhard-Karls-Universitaet Tuebingen, Tuebingen, Germany (WV)

Correspondence to: Livaniou Evangelia, Immunopeptide Chemistry Laboratory, Institute of Radioisotopes and Radiodiagnostic Products, National Centre for Scientific Research "Demokritos," Aghia Paraskevi Attikis, Athens 15310, Greece. E-mail: livanlts{at}rrp.demokritos.gr

	Summary

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Prothymosin (ProT) is a nuclear polypeptide of great biological and, possibly clinical, importance, because its expression levels have been associated with early diagnosis/prognosis of human cancer. It is therefore interesting to raise easily available and cost-effective antibodies that would be applied to develop reliable ProT immunodiagnostics. In this study, New Zealand white rabbits and laying hens were parallel immunized against intact ProT or the synthetic fragments ProT[1-28], ProT[87-109], and ProT[101-109], all conjugated to keyhole limpet hemocyanin (KLH). The corresponding antibodies G and Y were immunochemically evaluated in parallel with ELISA and Western blot systems and applied to fluorescence immunocytology experiments using various cancer cell lines and normal cells. The antibody G raised against ProT[101-109]/KLH had excellent functional characteristics in the Western blot and immunocytology experiments, where the fluorescent signal was almost exclusively shown in the cell nucleus independently of the cells assayed. The above antibody has been applied to preliminary IHC staining of human cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinoma tissue; this antibody can be further used in cancer tissue immunostaining and in research concerning the role of ProT in tumorigenesis. (J Histochem Cytochem 56:1023–1031, 2008)

Key Words: prothymosin • polyclonal antibody • ELISA • Western blot • immunocytology • immunohistochemistry • cancer prostate tissues

	Introduction

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PROTHYMOSIN (ProT) is a nuclear polypeptide that was first isolated from the rat thymus gland (Haritos et al. 1984; Hannappel and Huff 2003). The primary structure of ProT is almost identical in all mammalian species (human ProT: 109 amino acids, molecular mass = 12.6 kDa) and shows some unusual features, such as the lack of aromatic amino acids; under physiological conditions, ProT adopts a random coil conformation, with no secondary structure (Gast et al. 1995).

Although its biological role has not been completely elucidated, literature points toward a dual role for ProT: an extracellular one, associated with cell-mediated immunity (Cordero et al. 1997; Skopeliti et al. 2006), and an intracellular one, related to cell proliferation and apoptosis (Bustelo et al. 1991; Rodriguez et al. 1998; Jiang et al. 2003). Increased intracellular expression of ProT, which has been thought to be an oncoprotein (Karapetian et al. 2005; Kobayashi et al. 2006), has been observed in several types of human cancer. Recently, several groups have applied IHC techniques and reported that ProT is overexpressed in various cancers, e.g., gastric (Leys et al. 2007), prostate (Suzuki et al. 2006), and thyroid (Letsas et al. 2005) cancer, and might therefore be considered as an intracellular tumor biomarker.

Elucidation of the putative diagnostic and/or prognostic significance of ProT in human cancer would be greatly facilitated by an easily available and cost-effective antibody for the polypeptide that could be further applied to develop reliable ProT–in vitro immunodiagnostics for routine use. On the other hand, before its application in disease immunodiagnosis, any antibody for ProT should be carefully characterized in terms of its specificity and its ability to recognize ProT fragments as well, because intact ProT might be processed inside the cell by various enzymes, such as asparaginyl endopeptidase (Sarandeses et al. 2003) or caspases (Enkemann et al. 2000a; Evstafieva et al. 2000,2003), leading to different intracellular ProT fragments that may retain ProT immunoreactivity but may differ in their biological functions from the intact polypeptide.

In this study, we developed different antibodies for ProT using different immunogens and different host animals. More specifically, we developed antibodies against intact, native ProT of bovine origin or against the synthetic fragments ProT [1-28], ProT[87-109], and ProT[101-109], all conjugated to the carrier protein keyhole limpet hemocyanin (KLH). The N-terminal fragment ProT[1-28] and the C-terminal fragments ProT[87-109] and ProT[101-109] were selected as antigens, because the antigenic determinants of a polypeptide molecule are usually located in its N and/or C termini. Moreover, the N-terminal fragment ProT[1-28] is identical to the bioactive peptide T1, which might be an endogenous biomolecule resulting from the intracellular proteolysis of ProT by asparaginyl endopeptidase (Sarandeses et al. 2003). On the other hand, the peptides ProT[87-109] and ProT[101-109] are located in the C-terminal area ProT[88-108], which presents relatively high hydrophilicity and mobility indices and is therefore likely to have high antigenicity, according to a previous theoretical study (Costopoulou et al. 1998). Another, practical reason for selecting these peptides as candidate antigens is that they both have a lysine residue in their N terminus, and it is therefore expected that, using the glutaraldehyde method (Avrameas 1969), they will be linked to the carrier protein KLH mainly through the N- and the N-amino groups of their N termini, thus leaving their C termini to be exposed unmodified to the B lymphocytes of the immunized animal.

The above immunogens were parallel administered to New Zealand white rabbits and laying hens, so that antibodies G and Y were developed, respectively. Laying hens were previously immunized against ProT/KLH (Klimentzou et al. 2006) in an effort to apply the so-called "IgY technology" (Schade et al. 2001) to ProT, because the avian immune system is expected to react better against a highly conserved mammalian polypeptide (Tini et al. 2002; Schade et al. 2005), such as ProT; in this study, a whole series of immunogens (i.e., ProT/KLH, ProT[1-28]/KLH, ProT[87-109]/KLH, ProT[101-109]/KLH) was used for raising the corresponding antibodies Y. The antibodies G and Y thus developed were parallel evaluated in ELISA and Western blot systems and then applied to fluorescence immunocytology experiments. The antibody showing the highest efficiency in Western blot and especially in fluorescence immunocytology (anti-ProT[101-109]/KLH) was further applied to preliminary IHC staining of human cancer prostate tissues.

	Materials and Methods

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Materials
ProT (generous gift of J. Czarnecki, PhD, Vienenburg, Germany, and M. Pesic, MD, Bad Harzburg, Germany) was isolated from bovine thymus. Thymosin β4 (Tβ4) was synthesized, purified, and characterized as previously described (Zikos et al. 2003). All laboratory solvents and chemicals were purchased from Merck (Darmstadt, Germany) or Sigma (St. Louis, MO) and they were analytical grade.

Peptide Synthesis
ProT[1-28] (=T1), ProT[87-109], and ProT[101-109] were synthesized by the Fmoc-solid phase peptide synthesis following a protocol that has been previously described (Zikos et al. 2003). The peptides synthesized were purified on a Waters 600E HPLC System (Waters; Milford, MA) by semipreparative reverse phase-high performance liquid chromatography (RP-HPLC) using a 250 x 12.7-mm (ID) Nucleosil 7 C18 column (Macherey Nagel; Dueren, Germany). A solvent system consisting of 0.05% trifluoroacetic acid (TFA) in water (Solvent A) and 60% CH₃CN in solvent A (Solvent B), a flow rate of 8 ml/min, and linear gradients from 100% A to 55% A in 52 min (ProT[1-28]) and from 100% A to 60% A in 47 min (ProT[87-109]) was applied. Crude ProT[101-109] was of high purity, and further purification was not necessary. The purified peptides were characterized by analytical RP-HPLC on a Waters 616 (996 PDA detector) HPLC system using a 250 x 4.6-mm (ID) LiChrospher RP C18 column (5-µm particle size; Merck). A solvent system consisting of 0.05% TFA in 0.1 M NaCl (Solvent A) and 0.05% TFA in CH₃CN (Solvent B), a flow rate of 1.0 ml/min, and linear gradients from 100% to 40% A in 19 min (ProT[1-28]) and from 100% to 80% A in 19 min (ProT[87-109], ProT[101-109]) was applied. Peaks were detected spectrophotometrically (220 nm). The purified peptides were also characterized by electrospray ionization-mass spectrometry (ESI-MS) on an Esquire3000plus ion trap mass spectrometer (Bruker-Daltonics; Bremen, Germany). Better overall yield and higher purity were obtained in the order ProT[101-109], ProT[87-109], ProT[1-28].

Immunization of Animals
New Zealand white rabbits were intradermally or subcutaneously injected at many sites on their back (Vaitukaitis 1981) with ProT, ProT[1-28], ProT[87-109], or ProT[101-109] conjugated to KLH (ProT/KLH, ProT[1-28]/KLH, ProT[87-109]/KLH, or ProT[101-109]/KLH). Conjugation was performed according to the glutaraldehyde method, as previously reported for the N-terminal fragment [1-14] of Tβ4 (Livaniou et al. 1992). The immunogens were injected as emulsions (1:1, v/v) in complete Freund's adjuvant. The animals were boosted initially after 6 weeks and subsequently every 4 weeks. Blood was collected 2 weeks after each booster injection. Antisera (source of antibodies G) were obtained with low-speed centrifugation of whole blood and stored at –35C.

Laying hens were immunized with ProT/KLH, ProT[1-28]/KLH, ProT[87-109]/KLH, or ProT[101-109]/KLH (same products as above) as previously described for ProT/KLH (Klimentzou et al. 2006). The antibodies Y were isolated from egg yolk as previously reported (Klimentzou et al. 2006). Care of the animals was in accordance with the corresponding European legislation.

Characterization of Antibodies With ProT-ELISAs
Titration of antibodies was performed in a titer-ELISA system as previously described (Costopoulou et al. 1998; Klimentzou et al. 2006). The ability of antibodies to discriminate between native ProT and synthetic fragments of it was performed by comparing the corresponding ELISA displacement curves, as previously described for antibody Y (Klimentzou et al. 2006).

Application of Antibodies in Western Blot Analysis of Cell and Nuclear Lysates
Cell Culture: Preparation of Total Cell and Nuclear Lysates
HeLa cells, PANC-1 cells, and human fetal fibroblasts were cultured in D-MEM (PAA Laboratories; Pasching, Austria), supplemented with 10% FCS (Biochrom KG; Berlin, Germany), at 37C in a 5% CO₂ incubator.

The culture medium was discarded, and the cell monolayers were washed with 0.9% NaCl. Trypsin/EDTA [0.05%/0.02% (w/v) in PBS (0.01 M PBS, pH 7.4) free of Ca²⁺ and Mg²⁺; Biochrom AG] was added, and the cells were incubated for 10 min at 37C. The cells were resuspended in 0.9% NaCl and centrifuged (1500 x g for 10 min). For preparing (total) cell lysates, the supernatant was discarded, and the cells were resuspended in single-detergent cell lysis buffer (0.05 M Tris-HCl, pH 8.0, 0.9% NaCl, 0.02% NaN₃, 100 µg/ml PMSF, 1 µg/ml aprotinin, 1% NP-40), incubated on ice for 10 min, and centrifuged (1500 x g for 10 min). The supernatant was transferred to a microtube and stored at 4C. For preparing nuclear lysates, after resuspension in 0.9% NaCl and centrifugation, the supernatant was discarded, and the cells were resuspended in hypotonic solution (0075 M KCl) and incubated on ice for 15 min. Samples of the suspension were checked on microscope to ensure that the cell membrane was disrupted and the suspension was centrifuged (1500 x g for 10 min). The supernatant was discarded, the cell nuclei were resuspended in the cell lysis buffer, and the above described procedure was followed.

Western Blot Analysis of Cell and Nuclear Lysates
Cell lysates (aliquots corresponding to 60 or 30 µg total protein), as well as nuclear lysates (60 or 30 µg total protein) from HeLa cells, PANC-1 cells, and human fetal fibroblasts, along with a ProT solution (10 ng) as positive control, were subjected to SDS-PAGE and electrotransferred on a nitrocellulose membrane (ProTran BA83; Schleicher and Schuell, Dassel, Germany), which was preactivated according to a previously described protocol (Papamarcaki and Tsolas 1994). Before the electrotransfer, the SDS-PAGE gel and the preactivated membrane were equilibrated for at least 90 min in the electrotransfer buffer (0.02 M CH₃COONa, pH 4.5). After electrotransfer, the membrane was blocked with blocking buffer A (1% BSA, 20% FCS, and 0.05% Tween 20 in PBS) for 1 hr at room temperature on a shaker, washed twice with PBS (for 5 min each time), and incubated with rabbit antibody G (anti-ProT/KLH, anti-ProT[1-28]/KLH, anti-ProT[87-109]/KLH or anti-ProT[101-109]/KLH antiserum, diluted 1:200 with diluting buffer A: 0.2% BSA, 2% FCS, and 0.05% Tween20 in PBS) or a solution of hen antibody Y (anti-ProT/KLH, 25 µg/ml in diluting buffer A) for 1 hr at room temperature. The membrane was washed three times with PBS (for 5 min each time) and incubated for 30 min at room temperature with goat anti-rabbit IgG/horseradish peroxidase (HRP; Sigma), diluted 1:2000 with diluting buffer A, or rabbit anti-hen IgY/HRP (Chemicon; Temecula, CA), diluted 1:3000 with diluting buffer A, respectively. The membrane was washed three times with PBS (for 5 min each time), and the protein bands were visualized with chemiluminescence (ECL kit; Amersham Biosciences, Uppsala, Sweden), according to the manufacturer's instructions.

Control SDS-PAGE gels were run in parallel with those used in the electrotransfer; protein bands in these gels were stained with Coomassie blue R250.

Application of Antibodies to Fluorescent Immunocytology
HeLa, PANC-1, MCF-7, MDA-MB-231 cells, and human fetal fibroblasts were grown on special object slides and fixed with 4% paraformaldehyde for 30 min at room temperature. The slides were washed twice with PBS (for 2 min each time), incubated with Triton X-100 (0.5% in PBS) for 10 min at room temperature, and washed three times with PBS (for 5 min each time). Blocking was performed by incubating with blocking buffer A for 1 hr at room temperature. The slides were washed three times with PBS (for 5 min each time) and incubated with rabbit antibody G (anti-ProT/KLH, anti-ProT[1-28]/KLH, anti-ProT[87-109]/KLH, or anti-ProT[101-109]/KLH antiserum, diluted 1:400 with diluting buffer A) or hen antibody Y (anti-ProT/KLH, 30 µg/ml, in diluting buffer A) for 1 hr at room temperature. The slides were washed three times with PBS (for 5 min each time) and incubated either with donkey anti-rabbit-IgG/Cy3 (Fa. Dianova; Hamburg, Germany), diluted 1:500 with diluting buffer A, for 1 hr at room temperature, or with rabbit anti-hen IgY/biotin (Chemicon), diluted 1:2500 with diluting buffer A, for 1 hr at room temperature and then, after washing, with streptavidin-FITC, 0.5 µg/ml in diluting buffer A, for 30 min at room temperature, respectively. The slides were washed three times with PBS (for 5 min each time), incubated with an appropriate solution for DNA staining [YOPRO and TOPRO, respectively (Molecular Probes; Leiden, The Netherlands), diluted 1:2000 and 1:500 with diluting buffer A] for 5 min at room temperature, and washed once more with PBS. Finally, they were mounted with MOWIOL (Calbiochem; San Diego, CA) and observed with a confocal microscope (Leica TCS SP; Leica Microsystems, Bensheim, Germany). Preimmune rabbit serum or hen immunoglobulins were used as a negative control instead of the specific antibody G or Y, respectively, at the same dilution. Moreover, anti-ProT[101-109]/KLH antiserum (1:400) preincubated (2 hr) with an aqueous solution of ProT (10 µg/ml, final volume) was used as a negative control in "blank" experiments.

Application of the Antibody G Against ProT[101-109]/KLH to IHC Staining of Cancer Prostate Tissues
Tissue sections (5 µm) cut from paraffin blocks were dewaxed by incubation in xylene for 30 min at 55–60C (waterbath) and then for 5 min at room temperature. After immersion in absolute ethanol, any endogenous peroxidase activity was blocked by incubation with a solution of 1% H₂O₂/30% MeOH for 30 min; the tissue sections were rehydrated by passing through a graded series of ethanol and water mixtures (96–70%, 2 min) and distilled water (5 min). Microwave pretreatment for reactivating antigenicity was carried out in 0.01 M citrate buffer, pH 6.0, at 180 (4 min), 360 (4 min), and 480 W (2 min). After treatment with blocking solution B (0.2% BSA and 20% FCS in PBS-T, i.e., 0.01% Triton X-100 in PBS), tissue sections were incubated with the anti-ProT[101-109]/KLH antiserum, diluted 1:100 with diluting solution B (0.2% BSA and 2% FCS in PBS-T), for 1 hr at room temperature, washed twice with PBS-T (for 5 min each time), and incubated with goat anti-rabbit IgG-biotin (Sigma), diluted 1:300 with diluting solution B, for 40 min at room temperature. Tissue sections were washed twice with PBS-T (for 5 min each time), incubated with streptavidin-HRP (Sigma), 1 µg/ml in PBS for 40 min at room temperature, and washed twice with PBS (for 5 min each time), followed by the addition of the chromogen (diaminobenzidine; Sigma). Finally, the slides were counterstained with Harry's hematoxylin, dehydrated by passing through a graded series of ethanol and water mixtures (70%, 96%, absolute ethanol, 2 min), cleared with xylene (2 min), and mounted with coverslips using Entellan. Preimmune serum, diluted 1:100 with diluting solution, as well anti-ProT[101-109]/KLH antiserum (1:100) preincubated (2 hr) with an aqueous solution of ProT (10 µg/ml final volume), were used as negative controls in "blank" experiments.

	Results

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Characterization of Antibodies With ProT-ELISAs
Best titers were obtained with the antibody G against ProT[1-28]/KLH and ProT[101-109]/KLH (titer of the corresponding antisera: 1:7500). The anti-ProT/KLH and anti-ProT[87-109] antisera gave lower titers, 1:4000 and 1:1500, respectively. The anti-ProT/KLH antibody Y gave a titer of 1.7 µg/ml, whereas the anti-ProT[1-28]/KLH, anti-ProT[87-109]/KLH, and anti-ProT[101-109]/KLH antibody Y gave a very low ratio of specific to background signal, and no titer value could be estimated for them.

The anti-ProT[101-109]/KLH antibody G could recognize native ProT and both of the C-terminal synthetic fragments tested, whereas it did not recognize T1 (Figure 1 ). Similar results were obtained with the anti-ProT[87-109]/KLH antibody G. The antibody G against ProT[1-28]/KLH could recognize both native ProT and the N-terminal synthetic fragment but could not recognize—as expected—the C-terminal synthetic fragments. The antibodies G and Y raised against ProT/KLH gave similar results with those previously reported (Klimentzou et al. 2006). As expected, none of the above antibodies cross-reacted with structurally irrelevant peptides, such as Tβ4, a peptide of the β-thymosin family.

View larger version (10K): [in this window] [in a new window]   Figure 1 ELISA displacement curves obtained with the anti-prothymosin (ProT)[101-109]/keyhole limpet hemocyanin (KLH) antibody G in the presence of increasing concentrations (100 ng/ml to 100 µg/ml) of ProT, ProT[1-28], ProT[87-109], and ProT[101-109]. Symbols corresponding to the intact polypeptide and the different synthetic fragments are shown as an inset.

View larger version (10K): [in this window] [in a new window]   Figure 2 Western blot analysis of cell and nuclear lysates from HeLa cells, PANC-1 cells, and human fetal fibroblasts: PANC-1 cells, cell lysate, 60 µg total protein (Lane 1); PANC-1 cells, cell lysate, 30 µg total protein (Lane 2); HeLa cells, cell lysate, 60 µg total protein (Lane 3); HeLa cells, cell lysate, 30 µg total protein (Lane 4); human fetal fibroblasts, cell lysate, 30 µg total protein (Lane 5); ProT, 10 ng (Lane 6); PANC-1 cells, nuclear lysate, 30 µg total protein (Lane 7); HeLa cells, nuclear lysate, 60 µg total protein (Lane 8).

View larger version (26K): [in this window] [in a new window]   Figure 3 Immunolocalization of ProT in HeLa (A) and PANC-1 (B) cells. Dual-labeling (A1,B1), labeling for DNA (A2,B2), and specific immunolabeling for ProT with the anti-ProT[101-109] antibody G (A3,B3) are presented. As clearly observed, ProT-like immunoreactivity is localized almost exclusively in the cell nucleus.

Application of the Antibody G Against ProT[101-109]/KLH to IHC Staining of Cancer Prostate Tissues

The basal and luminal cells of normal and hyperplastic glands were positive with diffuse, but alternating, nuclear positivity. Stain intensity of the malignant epithelial cell nuclei looked similar to that of the non-neoplastic epithelium. In all carcinomas, ProT expression was observed in >50% of the cells in continuity, regardless of Gleason's grading (Figures 4A and 4B).

View larger version (135K): [in this window] [in a new window]   Figure 4 (A) ProT-negative normal prostatic gland adjacent to strongly positive adenocarcinoma cells. (B) Diffuse strong ProT positivity of both adenocarcinoma and prostatic intraepithelial neoplasia lesions. (C) Prostatic tissue stained with anti-ProT[101-109]/KLH antibody G. (D) Negative control, i.e., prostatic tissue stained with anti-ProT[101-109]/KLH antibody G preincubated with ProT.

	Discussion

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Intact ProT and appropriately selected synthetic fragments of the molecule were conjugated to KLH and administered to rabbits and hens to develop antibodies G and Y, respectively, following previously presented methodologies (Costopoulou et al. 1998; Klimentzou et al. 2006). Characterization of antibodies G and Y raised against ProT/KLH in ProT-ELISAs and dot-blots has been recently reported and commented on by our group (Klimentzou et al. 2006).

The antibody G raised against ProT[1-28]/KLH, ProT[87-109]/KLH, and ProT[101-109]/KLH, as those previously developed against similar immunogens (Costopoulou et al. 1998), showed moderate titers for the ProT molecule, which are, however, at least comparable to those thus far reported in the literature (Yialouris et al. 1988; Loidi et al. 1997). On the contrary, the antibody Y raised against ProT[1-28]/KLH, ProT[87-109]/KLH, and ProT[101-109]/KLH showed a very low specific signal during titer determination. This experimental result, which indicates a further "limit" in our efforts to successfully apply the "IgY technology" to the development of antibodies with the highest possible titer and specificity for ProT (Klimentzou et al. 2006), might be associated with differences in the ProT epitopes that are recognized by hen and rabbit B lymphocytes (Carlander et al. 1999). This assumption is supported by our findings according to which the antibody Y raised against ProT/KLH recognizes, to a much lesser extent, the N-terminal fragment ProT[1-28] than the antibody G raised against the same immunogen (Klimentzou et al. 2006).

The antibody G against ProT/KLH, ProT[1-28]/KLH, ProT[87-109]/KLH, and ProT[101-109]/KLH (as well as the antibody Y against ProT/KLH) gave similar ELISA displacement curves, ranging roughly from 0.1 to 100 µg/ml, in the presence of increasing concentrations of ProT in solution (Figure 1). In general, mainly because of their moderate titer values, such antibodies are difficult to apply to the development of sensitive immunoassay systems, at least those of the competitive type, in which they should be used at limited amounts. Affinity chromatography purification will not necessarily improve the antibody functional characteristics, because very often critical immunochemical features of low titer antibody molecules are destroyed during the purification process. On the other hand, monoclonal antibodies are usually characterized by rather low affinity for the corresponding antigen, and consequently, they are also difficult to apply to the development of sensitive competitive immunoassays. This is probably true for the few monoclonals for ProT reported until now in the literature (Staehli et al. 1983; Sukhacheva et al. 2002); as a consequence, few immunoassays have been reported for thymosins and even fewer have been applied to the analysis of biological samples (Panneerselvam et al. 1987; Tsitsiloni et al. 1994; Costopoulou et al. 1998; Mitani et al. 2000), whereas their quantitative experimental results seem to vary and therefore have been approached with caution by some researchers (Enkemann et al. 2000b). However, provided that their specificity is appropriate, carefully selected antibodies for ProT may be of great value for certain clinical applications and especially for the immunostaining of tissue samples, in which they can be used in excess.

The antibody G against ProT/KLH, ProT[1-28]/KLH, ProT[87-109]/KLH, and ProT[101-109]/KLH and the antibody Y against ProT/KLH were evaluated in a Western blot system. Optimal specificity was obtained with the antibody G against ProT[101-109]/KLH; using this antibody, ProT was detected in aqueous solutions (positive control) at amounts as low as—at least—10 ng; moreover, ProT could be detected in cell and cell nuclear lysates of HeLa and PANC-1 cells.

It should be noted here that the behavior of ProT in Western blotting has been an issue of controversy among research groups for many years (Freire et al. 2002; Sukhacheva et al. 2002; Karetsou et al. 2004). According to previous results of ours (Klimentzou et al. 2006), by using antibodies G or Y against ProT/KLH, no band corresponding to the molecular mass of ProT (11 kDa) could be observed on the Western blot membrane, although a large number of different experimental protocols had been tried. However, by elongating the washing step of the SDS-PAGE gel and the pretreated membrane in the electrotransfer buffer to at least 90 min, so that complete equilibration was ensured before electrotransfer, we were able to visualize a band corresponding to 11 kDa, for the first time, in the frame of this study. We strongly believe that the ProT molecule does behave in a tricky way in the Western blot (Freire et al. 2002; Lal et al. 2005), and the slightest changes in the experimental conditions may greatly affect its immobilization onto the membrane, which is probably the main reason for the often reported poor Western blotting results. However, under strictly adjusted conditions and careful handling, one can test the specificity of antibodies for ProT with Western blot.

The antibody G against ProT/KLH, ProT[1-28]/KLH, ProT[87-109]/KLH, and ProT[101-109]/KLH (and the antibody Y against ProT/KLH) was also evaluated in fluorescence immunocytology experiments, in which immunostaining was observed with confocal microscopy. Excellent results, i.e., negligible background signal and high reproducibility, were obtained with the antibody G against ProT[101-109]/KLH. By using this antibody, ProT-like immunoreactivity was located almost exclusively in the cell nucleus, where it appeared punctuate but widely dispersed, as previously reported by Enkemann et al. (2000b), who used COS-1 and NIH3T3 cells transfected with genes of epitope-tagged ProT proteins in combination with antibodies against the epitope tags. The intensity of the fluorescent signal was stronger in the cancer cells than in normal ones. Among the cancer cell lines studied, the strongest signal was observed in HeLa cells. This empirical observation was confirmed by the Western blot results. Nuclear localization of the immunostaining indicates that neither intact ProT nor shorter C-terminal fragments of it are present in the cell cytoplasm, at least not at detectable levels under the experimental conditions used, whereas the presence of T1 in the cytoplasm cannot be excluded, because the antibody used does not cross-react with T1. On the other hand, it would be interesting to study the immunostaining pattern obtained with a larger number of cancer cell lines, differing, for example, in their invasion potential.

The antibody G against ProT[101-109]/KLH, which gave the best results in the fluorescence immunocytology experiments, was further used in preliminary IHC experiments. According to the literature, IHC studies of ProT have been mainly performed by using antibodies raised against T1 (Dominguez et al. 1993), which may recognize both intact ProT and T1, thus raising a priori a "specificity issue." On the other hand, few IHC studies were performed with antibodies raised against C-terminal fragments of ProT, including the fragment ProT[101-109] (Costopoulou et al. 1997; Letsas et al. 2005); however, these studies have not provided pre-evaluation data on the specificity of the antiserum used with Western blotting or with single cell immunostaining.

In the frame of this study, the antibody G against ProT[101-109]/KLH was used for the localization of ProT in 15 prostate tissue sections obtained from patients undergoing total surgical prostatectomy. Before this, extensive negative control immunostaining was performed, in which anti-ProT[101-109]/KLH antiserum preincubated with ProT had been used, which confirmed the specificity of the antiserum (Figures 4C and 4D). Similar experiments with equally good results were also performed by immunocytology (data not shown).

In the only article—to our knowledge—reporting on the immunochemical localization of ProT in prostatic tissue samples, Suzuki et al. (2006) studied a large number of normal prostate, benign prostatic hyperplasia, and prostate cancer samples. According to the results of that study, ProT-like immunoreactivity was observed mainly in the cell nucleus, whereas a weak cytoplasmic staining was also observed; this may be attributed to the antibody the authors used (commercially available monoclonal antibody to ProT, clone 2F11), which, if identical to the one first described by Sukhacheva et al. (2002), recognizes an epitope on the N terminus of the ProT molecule (aa 1–31) and might, therefore, bind to cytoplasmic T1 as well. In addition, according to Suzuki et al. (2006), ProT-like immunoreactivity increased from normal epithelium to adenocarcinomas and was positively correlated with Gleason's grade as well as with disease clinical stage. In our study, strong nuclear immunostaining was observed in the malignant cells of all adenocarcinomas. The intensity of the staining was similar to that of adjacent hyperplastic glands, but the percentage of positive nuclei was higher in adenocarcinomas. Nevertheless, because of the small number of samples tested, this study cannot directly support, at least at this time, that ProT-like immunoreactivity increases in prostate cancer tissue compared with the adjacent benign prostate hyperplasia areas.

In summary, in this study, various antibodies G and Y for ProT were developed and evaluated in an attempt to produce an easily available and cost-effective antibody of the highest possible specificity for the polypeptide. According to the results obtained, the antibody G against ProT[101-109]/KLH, which could recognize intact ProT and C-terminal fragments of the molecule and did not recognize T1, showed excellent specificity and overall efficiency in Western blot and fluorescence immunocytology experiments. This antibody was applied to the IHC staining of a small number of cancer prostate tissues, leading to a high percentage of clearly and intensively stained nuclei in the adenocarcinomas. This well-characterized, easily available, and cost-effective polyclonal antibody can be further used in cancer tissue immunostaining, aiming eventually at both the establishment of a reliable ProT immunodiagnostics technique for potential routine use and the study of the role of prothymosin in tumorigenesis.

	Acknowledgments

 
This work was financially supported by the Programme "Excellence in the Research Institutes supervised by the Greek General Secretariat for Research and Technology."

The authors thank R. Nordin for excellent technical assistance in the immunocytology experiments, Dr. C. Zikos for advice concerning peptide synthesis, and Dr. A. Beck for the ESI-MS analyses. They also thank Dr. O. Tsitsilonis, Dr. H. Kalbacher, and especially Dr. G.P. Evangelatos for useful discussions, helpful suggestions, and ideas.

	Footnotes

 
Received for publication January 23, 2008; accepted August 1, 2008

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