Journal of Histochemistry and Cytochemistry, Vol. 49, 1537-1546, December 2001, Copyright © 2001, The Histochemical Society, Inc.


ARTICLE

Characterization of a Polyclonal Antibody to Human Pituitary Tumor Transforming Gene 1 (PTTG1) Protein

Sham S. Kakara, Leilei Chena, Rashmi Puria, Shawn E. Flynna, and Lothar Jennesb
a James Graham Brown Cancer Center, University of Louisville, Louisville, Kentucky
b Department of Anatomy/Neurobiology, University of Kentucky, Lexington, Kentucky

Correspondence to: Sham S. Kakar, Dept. of Medicine, 570 South Preston Street, Baxter Biomedical Research Building, Room 204 C, U. of Louisville, Louisville, KY 40202. E-mail: sskaka01@louisville.edu


  Summary
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Pituitary tumor transforming gene 1 (PTTG1), recently cloned from human testis, is a potent oncogene that is expressed in most tumors. However, assessment of its potential value as a prognostic marker is dependent on the development of a suitable antibody. We have developed a rabbit polyclonal antibody, SK601, that is highly specific for the PTTG1 gene product using recombinant PTTG1 protein (24 kD) containing an N-terminal His6 tag as the immunogen. The antiserum is capable of detecting recombinant PTTG1 protein in ELISA assays at a titer of 1:100,000. Use of the antibody as the probe in Western blotting analyses revealed a single band with the anticipated relative molecular weights of 52 kD from E. coli expressing the GST-PTTG1 recombinant protein, and 56 kD from COS-7 cells transfected with the PTTG1–GFP chimeric construct. A single band with a relative molecular weight of 28 kD was observed in extract of COS-7 cells transfected with PTTG1 cDNA. The antiserum immunoprecipitated a protein of relative molecular weight of 56 kD from the extracts of COS-7 cells transfected with the PTTG1–GFP chimeric construct. Immunohistochemical analysis of COS-7 cells transfected with this construct confirmed that the antibody detected and was specific for expressing the PTTG1–GFP recombinant protein. Screening of various normal human tissues (testis, ovary, and breast) by immunohistochemistry indicated that these tissues did not exhibit staining with the exception of testis, a tissue that had previously been shown to express PTTG1 mRNA. In contrast all of the tumor tissues (testicular tumor, ovarian tumor, and breast tumor) that were assessed exhibited intense staining. The results suggest that antiserum SK601 is highly specific for the PTTG1 protein and therefore should prove useful in further analysis of the expression and interactions of this protein, including its potential application as an immunohistochemical marker of human tumors. (J Histochem Cytochem 49:1537–1545, 2001)

Key Words: PTTG1, cancer, transforming gene, antibody


  Introduction
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Pituitary tumor transforming gene 1 (PTTG1), which was recently cloned from human testis in our laboratory, encodes a protein of 202 amino acids with no significant similarity to other known proteins (Kakar and Jennes 1998 ). Our initial studies were based on the work of Pei and Melmed 1997 , who used an mRNA differential display technique to clone PTTG from a rat pituitary tumor. Overexpression of PTTG1 in NIH 3T3 cells results in an increase in cell proliferation, induces cell transformation in vitro, and promotes tumor formation in nude mice (Pei and Melmed 1997 ; Kakar and Jennes 1998 ). PTTG1 mRNA is expressed at high levels in various human tumors, including tumors of the pituitary gland, adrenal gland, thyroid gland, liver, kidney, endometrium, uterus, ovary, breast, testes, and colon (Pei and Melmed 1997 ; Dominguez et al. 1998 ; Kakar and Jennes 1998 ; Lee et al. 1999 ; Zhang et al. 1999a , Zhang et al. 1999b ; Heaney et al. 2000a , Heaney et al. 2000b ; Puri et al. 2001 ). In contrast, the levels of PTTG1 mRNA in normal tissues are either very low or undetectable, with the exception of the testis. Studies of PTTG1 cloned from various ovarian tumors did not reveal any mutations in gene sequences, suggesting that the primary structure of PTTG1 is unaltered in ovarian tumors (Puri et al. 2001 ). Therefore, the overexpression of PTTG1 in tumorigenesis in human tumors is most likely associated with overexpression of the PTTG1 gene rather than accumulation of the altered PTTG1 gene product as a result of mutation.

In recent studies, Heaney et al. 1999 , Heaney et al. 2000a , Heaney et al. 2000b reported that high levels of PTTG1 protein in patients with colorectal and thyroid cancers are correlated positively with cell cycle and tumor metastasis and invasion. Ramos-Morales et al. 2000 have reported high levels of expression of PTTG1 in proliferating cells. Taken together, these reports suggest that PTTG1 may be important as a prognostic molecular marker in a variety of cancers. We therefore developed and characterized a polyclonal antibody that could be used for analysis of the PTTG1 gene product. Ideally, such a polyclonal antibody should be applicable in the immunocytochemical localization of the PTTG1 protein in normal and malignant tissues and proliferating cells, immunoprecipitation of the PTTG1 protein expressed in cultured cells in which studies of the PTTG modification, such as phosphorylation or mutagenesis, are being conducted, identification of the protein(s) that interacts with PTTG1 protein to mediate its cellular functions, and for clinical purposes to detect the level of PTTG1 protein expression in tumor tissues.


  Materials and Methods
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Construction of Plasmids
The human PTTG1 cDNA containing the His6 Tag at the 5' end was generated by using the specific primers and PTTG1 cDNA as a template in PCR (Kakar 1999 ). The primers used were sense 5'-ATACATATGCATCATCATCATCATCACATGGCTACTCTGATCTAT-3' (nucleotides 1–18) and antisense 5'-TCAGAATTCCACACAAACTCTGAAGCACT-3' (nucleotides 617–636). The sense primer contained the sequences for NdeI restriction enzyme and 6XHis. The antisense primer contained the sequence for the ECoRI restriction enzyme. The PCR conditions were initial denaturation at 95C for 5 min, then denaturation at 95C for 1 min, annealing at 54C for 1 min, and extension at 72C for 1 min for 30 cycles, with the final extension at 72C for 7 min after the last cycle of amplification. The PCR-generated DNA was restricted with NdeI and ECoRI restriction enzymes and subcloned into isopropyl-ß-D-thiogalactoside (IPTG)-inducible bacterial expression vector pET21b (Novagen; Madison, WI) at NdeI and ECoRI sites. The pET–PTTG1 cDNA was transformed into E. coli BL21 (D3) (Novagen) and plated on LB agar plates containing ampicillin (50 µg/ml). After IPTG induction, the construct (pET–PTTG1) expressed His6–PTTG1 protein containing a 6XHis tag at the N-terminal of PTTG1 protein. Ten colonies were selected by small-scale IPTG induction for the presence of a 24-kD recombinant protein on Coomassie Blue-stained SDS-PAGE. The clone showing the strongest induction was selected for large-scale PTTG1 protein purification.

The PTTG1–GFP chimeric construct was prepared by subcloning the full-length PTTG1 cDNA into pEGFPN3 (Clontech Laboratories; Palo Alto, CA) at the N-terminal of the GFP sequence at ECoRI and BamHI restriction sites. The PTTG1–pcDNA3.1 (PTTG1 cDNA) plasmid was prepared by subcloning the full-length PTTG1 cDNA into pcDNA3.1 plasmid (Invitrogen; Carlsbad, CA) at BamHI and ECoRI sites. The recombinant plasmids were selected by colony hybridization (Kakar 1999 ). The authenticity of the sequences and the orientation were determined by sequencing.

Production and Purification of PTTG Protein
The pET–PTTG1-transformed cells (BL21) were grown in LB medium containing ampicillin (50 µg/ml) with vigorous shaking at 37C until an OD600 of 0.5 was attained, and then incubated with 1 mM IPTG for 2 hr. Cells were harvested by centrifugation (10,000 rpm for 10 min) and the pellets were resuspended in lysis Buffer A [50 mM Tris, pH 8.6, 100 mM KCl, 0.1%. Nonidet P-40, 1 mM phenylmethyl sulfonyl fluoride (PMSF) and 1 µg/ml each of pepstatin, leupeptin, and aprotinin]. After sonication and centrifugation at 10,000 rpm for 10 min, the supernatant was incubated with Ni-NTA agarose beads at 4C for 60 min according to the manufacturer's instructions (Qiagen; Valencia, CA). The beads were washed with washing Buffer B (50 mM Tris, pH 8.6, 200 mM KCl, 0.1% Nonidet P-40, 1 mM PMSF, and 1 µg/ml each of pepstatin, leupeptin, and aprotinin) until the OD280 of the eluate was <0.01. The beads were transferred to a column and bound protein was eluted with a linear gradient of imidazole (Buffer B containing imidazole from 0 mM to 200 mM) with 2-ml fractions being collected. The protein in the fractions was analyzed by SDS-PAGE with Coomassie Blue staining. Fractions containing the protein were pooled and concentrated. The estimated recovery of purified PTTG1 protein was 6 mg/liter of bacterial culture.

Immunizations
Immunization of rabbits was performed by Genome System (Research Genetics; Huntsville, AL). Purified PTTG1 protein was emulsified with complete Freund's adjuvant (50:50, v/v) and injected SC into two New Zealand rabbits after preimmune serum had been collected. Then the animals were reimmunized and bled every 2 weeks until a high antibody titer was obtained.

Cell Culture and Transfections
COS-7 cell line was obtained from American Type Culture Collection (ATCC) (Manassas, VA) and was maintained at 37C in a humidified atmosphere of 5% CO2 in DMEM supplemented with penicillin/streptomycin (100 IU/ml and 100 µg/ml, respectively) and 10% fetal calf serum (HyClone, Atlanta, GA). For transient transfection approximately 24 hr before transfection, cells were seeded into six-well plates. Cells were transfected in serum-free conditions with 1.2 µg of plasmid DNA using lipofectamin reagent (Life Technologies; Gaithersburg, MD) according to the manufacturer's instructions. After 6 hr of transfection, the medium was replaced with regular growth medium (DMEM medium containing 10% FBS). After 48 hr of transfection the cells were harvested.

Western Blotting
Cells transfected with the appropriate construct were rinsed with PBS buffer, scraped off the plates, and pelleted by centrifugation. The cell pellets were resuspended in lysis buffer (20 mM Tris, pH 7.5, 1 mM EDTA, 0.1% Nonidet P-40, 1 mM PMSF, and 1 µg/ml each of pepstatin, leupeptin, and aprotinin) and lysed by sonication for 1 min. The total amounts of protein were determined by the method of Bradford (BioRad; Hercules, CA). A total of 20 µg of protein from each sample was then mixed with 2 x sample buffer, denatured at 95C for 3 min, and separated on 12% SDS-PAGE. Proteins were transferred to a nitrocellulose membrane (Amersham; Piscataway, NJ) using standard techniques. After transfer, the blots were stained with Ponceau S (Sigma; St Louis, MO) to confirm equal loading. Membranes were blocked with 5% nonfat dry milk in TBS/T (TBS with 0.1% Tween-20) for 1 hr and then incubated for 1 hr with anti-PTTG1 antiserum SK601 diluted 1:1500. The membranes were washed three times with TBS/T and then incubated for 1 hr with 1:5000 dilution of horseradish peroxidase-conjugated anti-rabbit antibody (Amersham). The bound antibody complexes were detected using the enhanced chemiluminescence (ECL) system from Amersham.

Immunohistochemical Analysis of the COS-7 Cells
COS-7 cells were transfected with pEGFPN3 vector or the PTTG1–GFP chimeric construct as described above. After 24 hr of transfection the cells were trypsinized, plated on Super-Frost Plus glass slides (Fisher Scientific; Springfield, NJ), and incubated at 37C. Cells were fixed with 4% freshly prepared paraformaldehyde for 8 min and then treated with 0.1% Nonidet P-40 for 5 min. Cells were pretreated with 5% normal goat serum for 60 min to block nonspecific binding sites and then were incubated at room temperature (RT) for 60 min with antiserum SK601 diluted 1 to 1,500. Control samples were incubated with preimmune serum. After several rinses with PBS buffer, sections were incubated with Texas Red-conjugated anti-rabbit secondary antibody for 45 min. (diluted 1:100) obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Cells were analyzed using a fluorescent microscope (Olympus X-70).

Immunohistochemical Analysis of the Human Tissues
Normal and tumor tissues were obtained from the Tissue Procurement Facility of the Comprehensive Cancer Center, University of Alabama at Birmingham. The tissues were collected at the time of biopsy or autopsy (3–12 hr after death) and were immediately frozen in liquid nitrogen and then stored at -80C. All human tissue specimens were obtained and analyzed with approval from University of Alabama at Birmingham and University of Louisville Human Studies Review Boards. The tissue sections 12-µm thick were cut and mounted on Super-Frost Plus glass slides and stored at -80C.

For immunohistochemical analysis, frozen tissue sections (12 µm) were thawed at RT. Endogenous peroxidase activity was blocked with 0.3% H2O2 in methanol for 20 min. The sections were incubated at 95C in 0.01 mol/liter sodium citrate buffer (pH 6.0) for 20 min to optimize antigen retrieval. Sections were pretreated with 5% normal goat serum for 60 min to block nonspecific binding sites and were incubated overnight at 4C with antiserum SK601 diluted 1:1500. Control sections were incubated with preimmune serum. After several rinses with PBS buffer, sections were incubated with biotinylated anti-rabbit secondary antibody for 30 min at RT. The sections were rinsed with PBS buffer and incubated with streptavidin–horseradish peroxidase-conjugated anti-rabbit antibody (Vector Laboratories; Burlingame, CA) for 30 min. After washing three times with PBS, the antibody sites were visualized by incubating with diaminobenzidine tetrahydrochloride at RT until the desired intensity developed. The slides were rinsed in tapwater, counterstained with hematoxylin, and mounted. At least three specimens from each normal and tumor tissue were used.

Immunoprecipitation
COS-7 cells were grown in six-well plates and transfected transiently with the PTTG1–GFP chimeric construct or the pEGFPN3 plasmid as described above. After 48 hr of transfection the cells were lysed in lysis buffer (20 mM Tris, pH 7.4, 140 mM NaCl, 0.1% Nonidet P-40, 1 mM PMSF, 1 µg/ml each of pepstatin, leupeptin, and aprotinin). The lysate was clarified by centrifugation at 15,000 rpm for 10 min. The supernatant (0.5 mg protein) was then incubated with a 1:500 dilution of preimmune serum for 60 min. The extract was incubated with protein A–agarose at 4C for 30 min and then centrifuged. The supernatant was collected and incubated again with protein A–agarose at 4C for 30 min. After centrifugation the supernatant was incubated with antiserum SK601 or with a 1:1500 dilution of preimmune serum for 1 hr at 4C. The extracts were incubated with protein A–agarose for 1 hr at 4C and the beads were collected by centrifugation. The beads were washed three times in lysis buffer and bound protein was recovered for SDS-PAGE analysis by boiling for 2 min in SDS sample buffer. After electrophoretic transfer, the membrane was subjected to Western blotting using an anti-GFP monoclonal antibody (Clontech) diluted 1:100 as suggested by the supplier, with detection using the ECL system.


  Results
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Production of Polyclonal Antibody
For production of a suitable polyclonal antibody, the full-length PTTG1 protein was used for immunization. As shown in Fig 1, induction of E. coli with IPTG resulted in a high level of expression of a 24-kD protein corresponding to the molecular mass of His6–PTTG1. The cells from IPTG-induced cultures were solubilized and His6–PTTG1 protein was then purified by Ni2+ affinity chromatography using Ni-NTA beads. Peak A280 fractions eluted from the column were pooled and showed predominately a single 24-kD band corresponding to His6–PTTG1 (Fig 1). The protein was concentrated and then injected into two New Zealand White rabbits. The antisera collected from both the rabbits 8 weeks after the first injection were tested using an ELISA assay. The highest dilution detected by ELISA was 1:100,000 for both antisera (SK601 and SK602). The highest dilution of both antisera that could detect a 24-kD protein band on Western blotting analysis of an extract of E. coli induced with IPTG was 1:20,000, with no reactivity with the extract of uninduced cells (results not shown). Because both antisera performed similarly in the screening assays, the antiserum from one rabbit (SK601) was selected for subsequent characterization.



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Figure 1. SDS-PAGE analysis of PTTG1 protein expressed in E. coli. Ten µg of bacterial extract from induced and uninduced cells and purified His6–PTTG1 protein was applied on each lane. Lane 1, uninduced bacterial cells; Lane 2, bacterial cells induced with IPTG; Lane 3, purified His6–PTTG1 protein. Arrow indicates the 24-kD His6-tagged PTTG1 protein.

Antiserum SK601 Detects PTTG1 Protein on Western Blotting Analysis
The antiserum was prepared using a PTTG1 protein tagged with six histidine residues and therefore it is possible that it could react with the His6 tag rather than the PTTG1 protein. Western blotting analysis of an extract of IPTG-induced E. coli that expressed GST–PTTG1 recombinant protein revealed specific reactivity with a protein of relative molecular weight of 52 kD, with no reactivity with the extract from uninduced cells (Fig 2). This reactivity with the GST–PTTG1 protein indicates that antiserum SK601 detects the PTTG1 protein and not the tag.



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Figure 2. Western blotting analysis of the bacterial cells expressing GST–PTTG1 protein using SK601 antiserum. Cell extracts (1 µg per lane) were electrophoresed on 12% SDS-PAGE, blotted, and probed with SK601 antiserum at dilutions of 1:5000 and 1:20,000. Lanes 1, 3, 5, and 7, cells uninduced; Lanes 2, 4, 6, and 8, cells induced with IPTG. Lanes 1–4, preimmune serum diluted 1:5000 (Lanes 1 and 2) and 1:20,000 (Lanes 3 and 4). Lanes 5–8, SK601 antiserum diluted 1:5000 (Lanes 5 and 6) and 1:20,000 (Lanes 7 and 8). The bands of lower molecular weight detected by antiserum are the result of partial degradation of PTTG1 protein or truncated PTTG1 proteins produced in E. coli.

As an independent test of the identity of the PTTG1 protein, we transfected COS-7 cells with PTTG1 cDNA or a PTTG1–GFP chimeric construct. After 48 hr of transfection, cell extracts were prepared and analyzed by Western blotting. As shown in Fig 3, specific reactivity with a protein of the anticipated relative molecular weight of approximately 28 kD was detected in the extracts of COS-7 cells transfected with PTTG1 cDNA, and specific reactivity with a protein of the anticipated relative molecular weight of 56 kD (PTTG1 + GFP) was detected in extracts of COS-7 cells transfected with the PTTG1–GFP construct. These results were confirmed by use of a GFP protein-specific monoclonal antibody. This antibody detected a 56-kD protein in extracts of COS-7 cells transfected with the PTTG1–GFP chimeric construct but not in extracts of cells transfected with PTTG1 cDNA (results not shown). Taken together, these results indicate that antiserum SK601 is highly specific and can detect PTTG1 protein expressed in mammalian cells as well as that expressed in E. coli.



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Figure 3. Western blotting analysis of the COS-7 cells. COS-7 cells were transfected with either PTTG1 cDNA or the PTTG1–GFP construct. The cell extracts were electrophoresed on 12% SDS-PAGE, blotted, and probed with SK601 antiserum diluted 1:1500. Lanes 1 and 3, COS-7 cells transfected with PTTG1 cDNA; Lanes 2 and 4, COS-7 cells transfected with the PTTG1–GFP construct. A band of 28 kD relative molecular weight was detected from COS-7 cells transfected with PTTG1 cDNA (Lane 3) and a band of 56 kD was detected from COS-7 cells transfected with the PTTG1-GFP construct (Lane 4). No band was detected when preimmune serum (Lanes 1 and 2) was used.

Immunohistochemical Detection of PTTG1 in Transfected COS-7 Cells
To address the question as to whether antiserum SK601 could detect PTTG1 protein in cultured cells and to further confirm that the antiserum was in fact detecting PTTG1 protein, we performed immunohistochemical analysis of COS-7 cells transiently transfected with the PTTG1–GFP chimeric construct and COS-7 cells transfected with the pEGFPN3 plasmid vector. After 24 hr of transfection, the cells were trypsinized, plated on Super-Frost Plus glass slides, and incubated for 24 hr. After attachment to slides, the cells were fixed with freshly prepared 4% paraformaldehyde and subjected to immunohistochemical analysis. As shown in Fig 4, approximately 20% of the cells were transfected with the PTTG1–GFP construct (green color for GFP protein). Immunofluorescence staining of the fixed COS-7 cells transfected with the PTTG1–GFP construct with 1:1500 diluted antiserum SK601 gave a cytoplasmic signal that was absent with preimmune serum. The cells that were immunoreactive with PTTG1 antiserum were stained with a uniform intensity (red) and this staining occurred only in the cells showing GFP protein expression (green). Pre-absorption of the antibody with the purified PTTG1 protein completely abrogated the staining of COS-7 cells transfected with the PTTG1–GFP chimeric construct (results not shown). Control COS-7 cells transfected with the pEGFPN3 vector were not stained by the SK601antiserum (Fig 4).



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Figure 4. Immunocytochemical analysis of the COS-7 cells. COS-7 cells were transiently infected with either pEGFPN3 plasmid (A–D) or PTTG1-GFP construct (E–H). After 48 hr of transfection, the cells were subjected to fluorescence analysis with preimmune serum (A,B,E,F) and antiserum SK601 (C,D,G,H). (A,C,E,G) Cells showing green fluorescence (indicated by arrowheads). (B,D,E,H) Cells stained with Texas Red (indicated by arrows).

Immunoprecipitation of PTTG1 Protein from COS-7 Cells
The ability of the antiserum SK601 to immunoprecipitate PTTG1 protein from COS-7 cells transfected with the PTTG1–GFP chimeric construct was then tested. Cell lysates were prepared 48 hr after transfection and immunoprecipitated using antiserum SK601. The immunoprecipitated products were separated on SDS-PAGE and subjected to Western blotting analysis, using a monoclonal antibody specific for the GFP protein. As shown in Fig 5, a distinct protein that migrates with a relative molecular weight of 56 kD was observed on immuoprecipitates of extracts of COS-7 cells transfected with the PTTG1–GFP chimeric construct. Such a band was not detected after immunoprecipitation with preimmune serum or if pEGFPN3 plasmid-transfected COS-7 cells were analyzed.



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Figure 5. Immunoprecipitation of PTTG1 cells transfected with the PTTG1-GFP construct. COS-7 cells were transfected transiently with the PTTG1-GFP construct or pEGFPN3 plasmid for 48 hr. Cell lysate (0.5 mg) was used for immunoprecipitation with SK601 antiserum. The protein–antibody complex was electrophoresed on 12% SDS-PAGE, blotted, and the blot probed with a GFP-specific monoclonal antibody. Lanes 1 and 3, COS-7 cells transfected with the PTTG1–GFP construct; Lanes 2 and 4, COS-7 cells transfected with pEGFPN3 plasmid. Lanes 1 and 2, preimmune serum diluted 1:1500; Lanes 3 and 4, antiserum SK601 diluted 1:1500. A band of 56 kD (arrow) was detected from COS-7 cells transfected with PTTG1-GFP construct. No band was detected when preimmune serum (Lane 4) was used.

Immunohistochemical Analysis of Human Tumor Tisues
It is anticipated that antiserum SK601 will be used to assess the expression of PTTG1 protein in tumors. To determine the utility of antiserum in this application, we examined various human normal and tumor tissues by immunohistochemical analysis. As shown in Fig 6, intense staining was observed in normal testis. Testis is composed of many cell types, both proliferating and differentiating. Maturation of germ cells proceeds through several ordered stages, with the stage of maturation increasing from base of the seminiferous tubules towards the lumen. Abundant PTTG1 protein immunostaining was detected in spermatocytes and spermatids, whereas only weak immunostaining was observed in Leydig cells (Fig 6B). Normal ovarian and breast tissues showed only weak or no staining (Fig 7). In contrast, ovarian tumor (Fig 7D) and breast tumor (Fig 7H), as well as testicular tumor (Fig 6D), stained intensely. No staining or weak staining was observed in tissues when preimmune serum was used.



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Figure 6. Immunohistochemical analysis of the human testis and testicular tumor. (A,B) Normal testis; (C,D) testicular tumor. (A,C) Preimmune serum; (B,D) antiserum SK601 diluted 1:1500. The level of expression of PTTG1 protein was higher in the tumor than that observed in normal tissue.



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Figure 7. Immunohistochemical analysis of the human ovary, ovarian tumor, breast and breast tumor. (A,B) Normal ovary; (C,D) ovarian tumor; (E,F) normal breast; and (G,H) breast tumor. (A,C,E,G) Preimmune serum; (B,D,F,H) antiserum SK601 diluted 1:1500. The levels of expression of PTTG1 protein were higher in tumors than those observed in normal tissues.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

PTTG1 is a novel oncogene that was recently identified in human testis (Kakar and Jennes 1998 ). Based on Northern blotting analysis and RT/PCR analysis of the PTTG1 mRNA from various human normal and tumor tissues, we and others have shown high levels of expression of PTTG1 mRNA in all tumors analyzed to date. In normal tissues, PTTG1 expression is either low or undetectable, except for the testis (Kakar and Jennes 1998 ; Puri et al. 2001 ). Overexpression of PTTG1 resulted in an increase in cell proliferation, induction of cell transformation in vitro, and promotion of tumor formation in vivo (Pei and Melmed 1997 ; Kakar and Jennes 1998 ; Zou et al. 1999 ). The molecular mechanisms by which PTTG1 induces cell transformation remain unclear. In recent studies, Zou et al. 1999 reported that overexpression of PTTG1 causes inhibition of sister chromatid separation, which may lead to genetic instability and thereby result in tumorigenesis. Zhang et al. 1999a , Zhang et al. 1999b and Heaney et al. 1999 reported an increase in secretion and synthesis of bFGF on expression of PTTG1 in NIH 3T3 cells and transactivation (Wang and Melmed 2000 ), suggesting that the PTTG1 gene product is multifunctional.

The reports that the levels of expression of PTTG1 are altered with cell proliferation and different stages of tumorigenesis suggest that PTTG1 may prove useful as a molecular marker of human tumorigenesis. Although measurement of PTTG1 mRNA by RT-PCR and Northern blotting analysis has been employed for this purpose (Kakar and Jennes 1998 ; Zhang et al. 1999a , Zhang et al. 1999b ; Heaney et al. 2000b ), these techniques are not optimal because they are indirect and do not lend themselves to the analysis of large numbers of fixed or frozen tissues, which are the most readily available source of clinical tissues. Immunohistochemical techniques are advantageous in that they can be readily applied to a large number of clinical samples, with the added benefit that they reveal the localization of proteins in tumors. Although some antibodies for the PTTG1 protein are currently available, these have not been well characterized. The goal of this study was to generate and characterize a polyclonal antiserum that is highly specific for PTTG1 protein and can be used for variety of purposes.

For production of the polyclonal antibody, a highly purified recombinant PTTG1 protein was used as the immunogen (Fig 1). Injection of this PTTG1 protein into rabbits produced antiserum that detected the PTTG1 protein at a dilution of 1:100,000 on ELISA. The antiserum SK601 detected the PTTG1 protein specifically when this protein was expressed in E. coli, indicating that the antiserum is capable of recognizing the PTTG1 protein when expressed in a prokaryotic system (Fig 2). The specificity of the antiserum was confirmed by analysis of COS-7 cells transfected with the PTTG1–GFP chimeric construct using Western blotting, immunocytochemical, and immunoprecipitation techniques. Western blotting and immunoprecipitation analyses of cell extracts from COS-7 cells revealed a single protein with a relative molecular weight of 56 kD (Fig 3 and Fig 5) and immunocytochemical analysis of the COS-7 cells showed staining of cells that were transfected with the PTTG1–GFP chimeric construct (Fig 4), whereas pre-absorption of antibody with the purified PTTG1 protein abrogated the immunostaining. Taken together, these results strongly support the concept that antiserum SK601 is highly specific and recognizes PTTG1 protein in the denatured as well as the native form.

The utility of PTTG1 antiserum in immunohistochemical analysis was demonstrated using frozen human normal and tumor tissues. Intense staining was observed in testicular tissues, testicular tumor (Fig 6), ovarian tumor, and breast tumor tissues (Fig 7). No staining or weak staining was observed when normal ovarian and breast tissues were examined or when preimmune serum was used. In human testicular tissues, intense immunostaining was localized to the spermatocytes and spermatids, with only weak staining of the Leydig cells, suggesting a high level of expression of PTTG1 protein in spermatocytes and spermatids and relatively lower expression in the Leydig cells. These results are consistent with our previous findings (Puri et al. 2001 ), and with those of Pei 1999 , who showed high levels of expression of PTTG1 mRNA in spermatocytes and spermatids through the use of in situ hybridization. In the testicular tumor, ovarian tumor, and breast tumor specimens, the staining was intense and widely distributed (Fig 6 and Fig 7). These results confirm our previous findings that were generated using RT-PCR and in situ hybridization (Kakar and Jennes 1998 ; Puri et al. 2001 ). No staining or weak staining of normal ovarian and breast tissues was observed. These results suggest that the antiserum SK601 is highly specific for the PTTG1 protein and can detect PTTG1 protein in frozen tumor sections.

In summary, we have produced an antiserum that is highly specific for the PTTG1 protein and that can detect the PTTG1 protein in its denatured form on Western blotting analysis and its native form when expressed in either a heterologous system or as the endogenous protein of tumors. Taken together, our findings strongly suggest that this antiserum will be of use in the evaluation of PTTG1 as an immunohistochemical marker of cell proliferation and tumorigenesis.


  Acknowledgments

Supported by grant CA82511 from the National Cancer Institute.

We wish to thank Dr Fiona Hunter for editorial assistance and valuable comments and Ms Shruti Lakhlani for technical help.

Received for publication March 30, 2001; accepted July 5, 2001.


  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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