Structure, Expression, and Function of Human Pituitary Tumor-Transforming Gene (PTTG)

Xun Zhang, Gregory A. Horwitz, Toni R. Prezant, Alberto Valentini, Masahiro Nakashima, Marcello D. Bronstein and Shlomo Melmed

Cedars-Sinai Research Institute-UCLA School of Medicine (X.Z., G.A.H., T.R.P., A.V., M.N., S.M.) Los Angeles, California 90048
Neuroendocrine Unit (M.D.B.) Division of Functional Neurosurgery University of São Paulo Medical School São Paulo, SP, Brazil 01406-100


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Despite advances in characterizing the pathophysiology and genetics of pituitary tumors, molecular mechanisms of their pathogenesis are poorly understood. Recently, we isolated a transforming gene [pituitary tumor-transforming gene (PTTG)] from rat pituitary tumor cells. Here we describe the cloning of human PTTG, which is located on chromosome 5q33 and shares striking sequence homology with its rat counterpart. Northern analysis revealed PTTG expression in normal adult testis, thymus, colon, small intestine, brain, lung, and fetal liver, but most abundant levels of PTTG mRNA were observed in several carcinoma cell lines. Stable transfection of NIH 3T3 cells with human PTTG cDNA caused anchorage-independent transformation in vitro and induced in vivo tumor formation when transfectants were injected into athymic mice. Overexpression of PTTG in transfected NIH 3T3 cells also stimulated expression and secretion of basic fibroblast growth factor, a human pituitary tumor growth-regulating factor. A proline-rich region, which contains two PXXP motifs for the SH3 domain-binding site, was detected in the PTTG protein sequence. When these proline residues were changed by site-directed mutagenesis, PTTG in vitro transforming and in vivo tumor-inducing activity, as well as stimulation of basic fibroblast growth factor, was abrogated. These results indicate that human PTTG, a novel oncogene, may function through SH3-mediated signal transduction pathways and activation of growth factor(s).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tumorigenesis is a multistep process, involving activation of oncogenes, growth factors, and their receptors, or the inactivation of tumor suppressor genes (1). Abnormal gene expression in tumor cells is associated with several characteristics that differ from normal cells, such as cell differentiation (2), DNA repair (3), cell-cell communication (4, 5), cell-matrix interaction (6), tumor invasion, motility and metastasis (7, 8), angiogenesis (9), and apoptosis (10). Despite major advances in studying these characteristics, understanding tumorigenesis as a continuous, multifunctional process is still limited.

Pituitary tumors are well differentiated adenomas accounting for ~10% of intracranial neoplasms, and clinically silent pituitary microadenomas are encountered in up to 23% of unselected adult autopsies (11, 12). These monoclonal adenomas are either nonfunctioning and do not secrete pituitary trophic hormones or are functioning and secrete one or more hormones including PRL, GH, ACTH, or rarely, glycoprotein hormones. These hypersecretory syndromes are associated with hypogonadism, infertility, acromegaly, Cushing’s disease, or rarely, hyperthyroidism (13). Most pituitary tumors are histologically benign. True pituitary carcinomas are extremely rare, and documentation of distant metastasis is the sole diagnostic criterion for malignancy (14).

Pituitary tumor pathogenesis has been extensively studied (15). Several intrinsic mutations resulting in activation of tumor-promoting genes as well as inactivation of tumor suppressor genes have been described, including G protein (Gs{alpha}) mutations (16, 17), rarely occurring ras mutations in invasive tumors (18, 19), loss of heterozygosity involving the 11q13 region (20), loss of purine-binding factor gene (nm23) expression (21), and, in mouse models, disruption of RB (22) and cyclin-dependent kinase inhibitors (23, 24). However, only G protein mutations have reproducibly been identified in a subset of sporadic GH-secreting pituitary adenomas (25). The molecular etiology of these tumors remains elusive, and other mechanisms may be invoked in pituitary tumorigenesis. Recently, a novel pituitary tumor-transforming cDNA (PTTG) was isolated in our laboratory from rat GH4 pituitary tumor cells (26). Overexpression of rat PTTG resulted in cell transformation in vitro and induced in vivo tumor formation in athymic mice.

We now characterize the human pituitary tumor-transforming gene (PTTG). It is abundantly expressed in malignant tumor cells, as well as in some normal tissues, and potently transforms cells both in vitro and in vivo. Overexpression of PTTG in transfected NIH 3T3 cells increases basic FGF mRNA level as well as stimulates its secretion. As point mutations in a proline-rich region near the PTTG C terminus abrogate its transforming ability and block basic FGF production, PTTG function in tumorigenesis may involve intracellular SH3 signals and growth factor production.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular Cloning of Human PTTG
An open reading frame containing 609 bp was revealed in the positive clones obtained from human fetal liver cDNA library using a 0.6-kb rat PTTG cDNA as screening probe (Fig. 1bGo). The presence of an in-frame stop codon before the predicted initiation codon suggests that it is complete. The homology between DNA sequences of this open reading frame and the coding region of rat PTTG is 85%. Amino acid sequence comparison between the translated product of this cDNA and rat PTTG protein reveals 77% identity and 89% homology. Since all positive clones were sequenced and no other cDNA fragment with higher homology was detected in the library, it appears that these cDNA clones represent human homologs of rat PTTG.



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Figure 1. Human PTTG Genomic and cDNA Structure

a, Human PTTG genomic structure, with numbering according to the cDNA sequence in panel b. The translation initiation (ATG) and stop (TAA) codons in the cDNA are indicated. NT, Nontranslated region. b, Nucleotide sequence of human PTTG cDNA. The translation initiation and stop codons for the open reading frame are bold and underlined. The in-frame stop codon upstream of the initiation codon is bold and italicized. c, Amino acid sequence of PTTG protein deduced from the coding region of cDNA. The basic amino acid-rich domain is underlined, and the basic amino acid residues are bold and italicized. The proline-rich motifs are bold.

 
GenBank search did not reveal a known protein with structural similarity to PTTG. However, we detected a basic amino acid-rich region near the N terminus (from position 58 to 101, with 32% basic amino acid residues) and a proline-rich region near the C-terminus of human PTTG protein (Fig. 1cGo). This proline-rich region contains two PXXP motifs consistent with the previously identified SH3-binding site (27). The presence of these functional motifs suggests that human PTTG protein may be involved in SH3 domain-mediated signal transduction pathways (28, 29).

The human PTTG cDNA was used to screen a human genomic library, and positive genomic clones were subjected to sequence analysis. The results were compared with the cDNA sequence of human PTTG and revealed four introns within the coding region (Fig. 1aGo).

Chromosomal Localization of Human PTTG
DNA from the Stanford Human-Hamster G3 Radiation Hybrid Mapping Panel was used as template in PCR reactions with PTTG-specific primers. The amplified products were sequenced to confirm that they indeed contain human PTTG sequences. By electronic analysis at the Stanford Human Genome Center website, PTTG was localized to 21.01 centirads from the marker D5S2576 with a LOD score of 8.48. According to neighboring Genethon markers in the SCIENCE96 Transcription Map, this marker is most likely located within the interval 5q32–34 of chromosome 5.

This mapping result was further confirmed by fluorescence in situ hybridization (FISH), using a 16-kb human genomic fragment containing PTTG as a probe. The initial experiment resulted in specific labeling of the distal long arm of a group B chromosome, which was believed to be chromosome 5 based on its size, morphology, and banding pattern. In a second experiment, a probe previously mapped to 5q21 was cohybridized with PTTG, resulting in specific labeling of the middle and distal long arm of chromosome 5, respectively (Fig. 2aGo). Among a total of 80 metaphase cells analyzed, 63 exhibited specific labeling, showing that human PTTG is located at a position that is 84% the distance from the centromere to the telomere of chromosome arm 5q, an area corresponding to band 5q33 according to the International System for Human Cytogenetic Nomenclature 1995. Thus, the results concur and localize PTTG to 5q33 (Fig. 2bGo).



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Figure 2. Chromosomal Localization as Determined by FISH

a, Metaphase chromosomes hybridized with human PTTG probe (arrow a) and 5q21 control probe (arrow b). b, Ideogram illustrating the chromosomal position of human PTTG at 5q33 according to the International System for Human Cytogenetic Nomenclature 1995. Arrow indicates human PTTG position.

 
Tissue Distribution of Human PTTG mRNA
The expression pattern of human PTTG mRNA in normal human adult and fetal tissues and in several human carcinomas is depicted in Fig. 3Go. A strong mRNA signal of approximately 0.8 kb was detected in human fetal liver (Fig. 3bGo). In normal human adult tissues, abundant PTTG expression was evident in testis. Strong expression was also observed in thymus, and weak expression signals were seen in colon, small intestine, brain, placenta, and pancreas (Fig. 3aGo). Interestingly, when human malignant tumor cells were tested, PTTG was found to be highly expressed in all cell lines examined (Fig. 3cGo). PTTG mRNA was also detected in several human pituitary tumors, including nonfunctioning, PRL-secreting, and ACTH-secreting tumors (Fig. 3dGo). No mutations of the PTTG-coding region in tumors were detected by RT-PCR followed by sequence analysis (data not shown).



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Figure 3. Tissue Distribution of Human PTTG mRNA

Two micrograms of poly A+ RNA derived from the indicated tissues were loaded in each lane. Top panel shows result of Northern hybridization to PTTG cDNA probe, and bottom panel shows hybridization to ß-actin probe (control). a, Normal human adult tissues. b, Normal human fetal tissues. c, Human cancer cells. From left to right: promyelocytic leukemia HL-60; HeLa cell S3; chronic myelogenous leukemia K-562; lymphoblastic leukemia MOLT-4; Burkitt’s lymphoma Raji; colorectal adenocarcinoma SW480; lung carcinoma A549; melanoma G361. d, Pituitary tumors. NF, Nonfunctioning tumor; PRL, PRL-secreting tumor; ACTH, ACTH-secreting tumor.

 
Effects of Human PTTG Overexpression on Cell Transformation and Tumor Induction
Since a GeneBank search revealed no known proteins structurally similar to PTTG, its role in tumor formation is as yet unclear. However, the presence of a proline-rich region containing PXXP motifs near the C terminus of PTTG protein suggests that it may be involved in SH3-mediated intracellular signal transduction pathways. To explore the function of this domain and its relationship to PTTG-transforming ability, we constructed human PTTG mutants by PCR-based site-directed mutagenesis. The following amino acid residues in the SH3-binding motifs were mutated: P163A, P170L, P172A, and P173L. Mutant cDNA, as well as wild-type PTTG cDNA, was cloned into the mammalian expression vector under control of the cytomegalovirus (CMV) promoter and stably transfected into NIH 3T3 cells. Overexpression of wild-type and mutant PTTG in each transfected cell line was confirmed by Northern analysis, RT-PCR followed by direct sequence analysis, and Western blot (Fig. 4Go). The point mutations did not change protein expression or susceptibility to proteolysis, since PTTG protein with the same molecular weight was expressed in each transfected cell type at similar levels, as shown by Western blot (Fig. 4cGo). The transforming ability of these cells was tested in an anchorage-independent growth assay. We found that NIH 3T3 cells overexpressing PTTG formed large colonies (numbers ranged from 198 ± 6 to 267 ± 23 colonies per plate, mean ± SD) on soft agar, while control NIH 3T3 cells containing the same expression vector but lacking PTTG cDNA insert did not induce significant colony foci under the same conditions (22 ± 1 colonies per plate) (Table 1Go). In contrast, the number and size of colonies formed from NIH 3T3 cells expressing mutant PTTG were greatly reduced (ranging from 57 ± 6 to 60 ± 5 colonies per plate) (Table 1Go and Fig. 5Go). Furthermore, when these cells were injected subcutaneously into athymic nude mice, PTTG-overexpressing cells caused tumor formation within 2 weeks in all five injected animals (tumor weights ranged from 560 to 1000 mg) (Fig. 6Go). In five mice injected with control transfectants (expression vector alone), only one developed a much smaller tumor weighing only 100 mg. As expected, when cells expressing mutant PTTG were injected into nude mice, no tumor formation was observed even after 3 weeks of injection (Fig. 6Go), consistent with the results obtained in the anchorage-independent growth assay. These results thus demonstrate the in vitro transforming activity and in vivo tumor-inducing potential of human PTTG and also strongly suggest that signaling protein(s) containing SH3 domain(s) mediate PTTG action.



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Figure 4. Wild-Type and Mutant PTTG Expression in Transfected NIH 3T3 Cells

a, A representative Northern blot in which 20 µg total RNA from each cell line were used to hybridize with human PTTG cDNA probe (top panel) or ß-actin probe (bottom panel). b, A representative sequencing gel from RT-PCR followed by direct sequence analysis showing wild-type PTTG and mutant PTTG expression in a respective transfectant. The arrows point to nucleotide changes. A silent mutation (*) was introduced to obtain equal melting points for the different primers. c, A representative Western blot in which 40 µg protein extracted from each transfectant were analyzed using a purified anti-PTTG polyclonal antibody. C, Cell lines transfected with vector alone; WT, cell lines transfected with wild-type PTTG expression vector; M, cell lines transfected with mutant PTTG expression vector.

 

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Table 1. Colony Formation by PTTG-Expressing Cells in Soft Agar

 


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Figure 5. Colony Formation of NIH 3T3 Cells Transfected with Wild-Type and Mutant PTTG Expression Vector on Soft Agar

a, Cells transfected with vector pCI-neo alone. b, Cells transfected with vector pCI-neo containing wild-type PTTG cDNA. c, Cells transfected with vector pCI-neo containing PTTG cDNA mutated in the proline-rich region (P163A, P170L, P172A, and P173L). d, High magnification of colonies formed from cells transfected with vector pCI-neo containing wild-type PTTG cDNA.

 


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Figure 6. Tumor Formation Induced by Human PTTG-Expressing Cells in Nude Mice

Each mouse was injected subcutaneously with 3 x 105 control, wild-type PTTG-overexpressing, or mutant PTTG-overexpressing cells. After 2 weeks, mice were photographed and killed, and their tumors were excised and weighed. Vector, Cell line transfected with vectors only; wt PTTG, cell line transfected with wild-type human PTTG expression vectors; mPTTG, cell line transfected with mutant human PTTG expression vector; None, no detectable tumor. The mouse on the left was injected with control cells transfected with vector only. The mouse in the middle was injected with cells transfected with wild-type PTTG expression vector. The mouse on the right was injected with cells transfected with mutant PTTG expression vector.

 
Stimulation of Basic FGF Expression and Secretion by Human PTTG
As PTTG-expressing NIH 3T3 cells were able to induce solid tumor growth in vivo, PTTG may activate growth factor and/or angiogenesis pathways. To test this hypothesis, we examined the expression of two important angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), in the PTTG-transfected NIH 3T3 cells. Northern analysis showed that, although no difference in VEGF mRNA expression was found in control and PTTG-transfected cells (data not shown), bFGF mRNA levels in PTTG-transfected cells were increased compared with mock-transfected control cells (Fig. 7aGo). In the cells transfected with mutant PTTG, the abundance of bFGF mRNA transcripts was low, similar to that observed in the control cells.



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Figure 7. Human PTTG Stimulates bFGF Production

a, A representative Northern blot in which 20 µg total RNA from each cell line were used to hybridize with human bFGF cDNA probe (top panel) or ß-actin probe (bottom panel). b, bFGF concentration in conditioned medium derived from each PTTG transfectant cultured for 72 h as measured by ELISA. C, Cell lines transfected with vector alone; WT, cell lines transfected with wild-type PTTG expression vector; M, cell lines transfected with mutant PTTG (P163A, P170L, P172A, and P173L) expression vector.

 
To further confirm bFGF regulation by PTTG at the protein level, enzyme-linked immunosorbent assay (ELISA) was performed to examine bFGF levels in conditioned culture medium. As shown in Fig. 7bGo, bFGF levels were markedly higher in the conditioned medium collected from wild-type human PTTG transfectants cultured for 72 h than those from control and mutant transfectants. Differences of total protein concentrations in these cultures were less than 10%. Therefore, PTTG not only enhances bFGF mRNA levels, but also stimulates bFGF secretion. The PTTG proline-rich region containing two SH3-binding motifs appears critical for this function.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have isolated a human gene that shows a striking structural similarity (85% identity for cDNA and 89% similarity for amino acid sequences) to rat PTTG. Human PTTG is located on chromosome 5, at position 5q33. Like its rat counterpart, it induces cell transformation in vitro and in vivo.

In normal adult rat tissues, PTTG expression is restricted to the testis (26). The normal tissue distribution of human PTTG is also limited; however, it is not restricted to testis but is also expressed in thymus, colon, small intestine, and, weakly, in brain and placenta, indicating that it may also play a role in certain normal cellular functions. The expression of human PTTG in fetal liver, but not in adult liver, suggests that it is differentially regulated and possibly functions during development. Interestingly, PTTG is highly expressed in all carcinoma cell lines tested, suggesting that it is a common and important factor for most malignant tumor types.

The chromosomal location of human PTTG, 5q33, is associated with reports of recurrent neoplastic abnormalities, including myeloid leukemia, chronic myeloproliferative disorder, myelodysplastic syndrome, squamous cell carcinoma, and lipoma (30). Using RT-PCR and direct sequencing, we examined PTTG in several human carcinoma cell lines, including cervix carcinoma HeLa cell, choriocarcinomas JEG-3 and JAR, breast adenocarcinoma MCF-7, osteogenic sarcoma U-2 OS, hepatocellular carcinoma Hep 3B, lung carcinoma EY, and thyroid carcinoma TC-1. Although no mutation was detected within the PTTG coding region, PTTG expression levels were high in all carcinoma cell lines tested. Thus, putative mutations in the gene-regulatory regions may cause PTTG dysregulation, and enhanced PTTG expression may mediate neoplastic cell transformation. We also found that PTTG mRNA was expressed in several pituitary tumors, indicating that it may be involved in pituitary tumorigenesis. Interestingly, PTTG mRNA levels in benign pituitary tumors, in general, were much lower than in malignant carcinomas (31). Therefore, although it seems that tumor PTTG expression correlates with malignancy, further confirmation is needed.

The transforming ability and high level of PTTG expression in malignant tumors demonstrate its involvement in tumorigenesis, although mechanisms of PTTG action and its relationship to other oncogene or tumor suppressor gene products is yet unclear. The presence of a basic amino acid-rich region and a proline-rich region in PTTG protein provide insights regarding its function. Basic amino acid-rich domains have been suggested as a nuclear localization signal (31, 32, 33), although the subcellular localization of PTTG is still undetermined. The proline-rich region of the human PTTG protein contains two PXXP motifs, which are potential binding sites for SH3-domains (27), important mediators of intracellular signal transduction (28, 34). Several proteins have recently been identified to contain PXXP motifs and bind to SH3 domains, such as GDP/GTP exchange factor SOS (35, 36), protein kinases JNK and phosphatidylinositol 3-kinase (37, 38), and the Abl oncogene product (39, 40). We report here that point mutations in the PXXP motif of human PTTG abrogated its transforming and tumor-inducing activity, despite expression of PTTG mRNA and protein in these mutants. Thus, this PXXP motif is important for PTTG-mediated transformation. Alternatively, another mechanism for the function of this region could involve serine phosphorylation of adjacent regions (41).

Interestingly, PTTG induces bFGF production at both the mRNA and protein levels. bFGF is a major activating factor for angiogenesis (42, 43), a necessary process for the expansion of the primary tumor mass as well as tumor metastasis (9, 44, 45). This is also in concurrence with our finding that transfected cells overexpressing PTTG cause formation of solid tumors in nude mice. Increased bFGF expression has been reported in several human tumors, such as pancreatic carcinoma (46), endometrial adenocarcinoma (47), advanced renal cell carcinoma (48), primary breast cancer (49), and pituitary tumors (50), in which it is considered a stimulating growth factor (51). Considering that the bFGF receptor is a protein tyrosine kinase and that PTTG increases bFGF production, we propose that one of the mechanisms of tumorigenesis by PTTG involves an SH3 protein interacting with PTTG, stimulating gene transcription and secretion of bFGF, and resulting in cell transformation and tumor formation. This hypothesis is supported by our observation that mutations of the proline-rich region, the potential SH3-binding site, abrogate bFGF production as well as cell transformation. These results establish human PTTG as a potent tumor-promoting factor whose functions involve bFGF production and cell transformation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Library Screening
A human fetal liver cDNA library (CLONTECH, Palo Alto, CA) was screened as described (51), using a 0.6-kb radioactively labeled cDNA fragment containing the entire rat PTTG coding region as a probe. The cDNA inserts from positive clones were subcloned into plasmid pBluescript-SK (Stratagene, La Jolla, CA) and subjected to sequence analysis using a Sequenase kit (United States Biochemical Corp., Cleveland, OH) and SequeGel System (National Diagnostics, Atlanta, GA).

A human genomic library (Stratagene, La Jolla, CA) was screened according to the manufacturer’s protocol, using the radioactively labeled human cDNA clone containing the complete coding region as a probe. DNA from each positive phage clone was purified using Lambda DNA preparation kit (Qiagen, Valencia, CA) and sequenced with a thermocycle sequencing kit (Amersham, Arlington Heights, IL) and SequaGel System (National Diagnostics).

Determination of Human PTTG Chromosomal Location
Chromosomal localization of human PTTG was determined by PCR analysis of the Stanford Human-Hamster G3 Radiation Hybrid (RH) Mapping Panel (Research Genetics, Huntsville, AL) as well as by FISH. DNA from 83 samples in the G3 RH mapping panel were amplified in 20-µl reactions containing 50 ng DNA, 1.75 U Expand High Fidelity enzyme (Boehringer-Mannheim, Indianapolis, IN), 1x reaction buffer with 1.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates (dNTPs), and 300 nM human PTTG-specific primers, 5'-CTGCCT-CAGATGATGCCTATCCAG-3'and 5'-CAAGCTCTCTCTCCTCGTCAAGG-3'. The PCR reactions were performed for 35 cycles of 94 C, 15 sec; 60 C, 30 sec; 68 C, 2.5 min. PCR products were visualized in 1% agarose gels stained with ethidium bromide and scored as positive ("1") if a strong (~3.5-kb PCR product) was visualized, negative ("0") if the band was not visualized, or ambiguous if a weak band was observed. The same results were obtained when the panel was again amplified with GIBCO-BRL (Gaithersburg, MD) Taq DNA polymerase. These data were submitted electronically to the Stanford Human Genome Center website to determine linkage to previously mapped markers. The FISH was performed at GenomeSystem, Inc. (St. Louis, MO). Briefly, a 16-kb human genomic fragment containing PTTG was labeled with digoxigenin dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes. Specific hybridization signals were detected by incubating the hybridized slides in fluoresceinated antidigoxigenin antibodies followed by counterstaining with 4,6-diamidino-2-phenylindole (DAPI).

Northern Blot Analysis
RNA blots (CLONTECH, Palo Alto, CA) derived from normal human adult and fetal tissue, as well as from malignant tumor cell lines and fresh pituitary tumor specimens, were hybridized with a 0.7-kb radiolabeled human cDNA fragment containing the complete coding region. Northern blot analysis was performed using ExpressHyb Solution (CLONTECH) or QuickHyb Solution (Stratagene, La Jolla, CA) according to the manufacturer’s protocol.

Western Blot Analysis
An antirat PTTG polyclonal antibody was developed as described previously (26). This antibody was purified by a PTTG affinity column that couples 10 mg synthetic antigenic peptide with HiTrap N-hydroxysuccinimide-activated column (Pharmacia, Piscataway, NJ) according to manufacturer’s protocol. The affinity column was washed with 75 mM Tris-HCl, pH 8.0, until no protein appeared in the eluent. The purified antibody was eluted with 0.1 M glycine, 0.5 M NaOH, pH 2.7, and neutralized with each volume of 2 N Tris-HCl, pH 8.0. Western blot was performed as described previously (26) using this purified antibody (1:100 dilution).

Stable Transfection of Human PTTG into NIH 3T3 Cells
The complete coding region of human PTTG cDNA was subcloned in frame into mammalian expression vectors pBK-CMV (Stratagene) or pCI-neo (Promega, Madison, WI) and transfected into NIH 3T3 fibroblast cells with Lipofectamine (GIBCO-BRL) according to the manufacturer’s protocol. Twenty-four hours after transfection, cells were serially diluted and grown in selection medium containing 1 mg/ml G418 for 2 weeks. Individual clones were isolated and maintained in selection medium. Total RNA was isolated from human PTTG-transfected cell lines as well as from control cells in which the vector pBK-CMV or pCI-neo had been transfected. Overexpression of human PTTG in each transfected cell line was confirmed by Northern blotting.

Site-Directed Mutagenesis
Point mutations on the proline-rich domain of PTTG peptide were generated by PCR-based site-directed mutagenesis. Two synthetic oligonucleotides, 5'-GATGCTCTCCGCACTCTGGGAATCCAATCTG-3' and 5'-TTCACAAGTTGAGGGGCGCCCAGCTGAAACAG-3', which would cause amino acid changes P163A, P170L, P172A, and P173L, were used to amplify human PTTG cDNA cloned into pBlueScript-SK vector (Stratagene). The amplified cDNA containing these mutations was then cloned into pCI-neo (Promega) and used in stable transfection. Expression of mutated PTTG product in transfected cells was confirmed by Northern analysis and RT-PCR followed by direct sequence analysis.

In Vitro and in Vivo Transformation Assay
Control and human PTTG-transfected cells were tested for anchorage-independent growth in soft agar (53). Three milliliters of soft agar (20% 2x DMEM, 50% DMEM, 10% FBS, and 20% 2.5% agar, melted and mixed at 45 C) were added to 35-mm tissue culture dishes. Ten thousand cells were mixed with 1 ml soft agar and added to each dish. Cells were incubated for 2 weeks before colonies were counted and photographed. For in vivo transformation, 1 x 105 control or human PTTG-transfected cells were resuspended in 400 µl PBS and injected subcutaneously into nude mice (five mice for each group). After two weeks, animals were photographed and tumors were excised and weighed.

Hybridization with VEGF and bFGF cDNA Probes
The cDNA probes for VEGF and bFGF were generated by RT-PCR using specific primers according to the published sequences (54, 55). These cDNAs generated from PCR were cloned and confirmed by sequence analysis. Total RNAs from cultured cells were extracted using Trizol Reagent (GIBCO-BRL) and used in Northern analysis as described previously (56).

ELISA of bFGF in Conditioned Medium
bFGF concentration in cell culture medium was assayed using Quantikine HS Human FGF Basic Immunoasssay Kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol. Cells (1 x 105) were plated in 100-mm cell culture dishes. After 72 h, the culture medium was collected, and 1 ml was lyophilized and resuspended in 200 µl PBS for ELISA assay.


    ACKNOWLEDGMENTS
 
We thank Drs. S. G. Ren and X. Li for technical help.


    FOOTNOTES
 
Address requests for reprints to: Shlomo Melmed, Academic Affairs, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 2015, Los Angeles, California 90048. E-mail: melmed{at}csmc.edu

This work was supported by NIH Grant DK-50238 (S.M.), Institutional Training Grant DK-7682 (X.Z.), and the Doris Factor Molecular Endocrinology Laboratory.

Received for publication July 23, 1998. Revision received October 5, 1998. Accepted for publication October 8, 1998.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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