Isolation and Characterization of a Pituitary Tumor-Transforming Gene (PTTG)

Lin Pei and Shlomo Melmed1

Division of Endocrinology and Metabolism Cedars-Sinai Research Institute-UCLA School of Medicine Los Angeles, California 90048


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pathogenesis of tumor formation in the anterior pituitary has been intensively studied, but the common mechanism involved in pituitary cell transformation and tumorigenesis remains elusive. In this study, we used mRNA differential display PCR to identify mRNAs that are differentially expressed in rat pituitary tumor cells compared with normal pituitary tissue. An mRNA exclusively expressed in pituitary tumor but not in normal pituitary was characterized. Using this pituitary tumor-specific PCR product as a probe to screen a cDNA library constructed from rat pituitary tumor GH4 cells, a cDNA of 974 bp was isolated. This cDNA encodes a novel protein of 199 amino acids, which contains no well characterized functional motifs. The mRNA of this cDNA is detected in normal adult testis and in embryonic liver, where the transcript is about 300 bp shorter and expressed at a much lower level than that detected from pituitary tumor cells. Overexpression of this protein in mouse 3T3 fibroblasts shows that it inhibits cell proliferation and induces cell transformation in vitro. Injection of transfected 3T3 cells into athymic nude mice resulted in tumor formation within 3 weeks in all animals. These results indicate that pituitary tumor cells express a unique and potent transforming gene (PTTG), which may play a role in pituitary tumorigenesis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Anterior pituitary tumors are mostly benign hormone-secreting or nonfunctioning adenomas arising from a monoclonal expansion of a genetically mutated cell (1, 2, 3). The most well characterized oncogene in pituitary tumors is gsp, a constitutively active Gsa resulting from activating point mutations in this gene (4, 5). Gsa mutations occur in about 40% of GH-secreting tumors, and constitutively activated cAMP response element binding protein is also found in a subset of these tumors (6). Although point mutations of Ras oncogene (7, 8), loss of heterozygosity near the Rb locus on chromosome 13 (9, 10, 11, 12, 13, 14), and loss of heterozygosity on chromosome 11 (15, 16, 17) have been implicated in some pituitary tumors, the mechanism that causes pituitary cell transformation remains largely unknown.

To clarify the molecular mechanisms involved in pituitary tumorigenesis, we used differential display PCR (18, 19) to identify mRNAs differentially expressed in pituitary tumor cells. This technique has been successfully used to identify mdm2 oncogene amplification in murine uterine adenocarcinomas (20) and melanin-concentrating hormone in the hypothalamus of ob/ob mice (21). We show here isolation and characterization of a pituitary tumor-derived gene (PTTG)2 that induces cell transformation in vitro and tumor formation in nude mice.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Molecular Cloning of Pituitary Tumor-Transforming Gene (PTTG) from Rat Pituitary Tumor Cells
Using 120 pair primers in the PCR, 11 DNA bands that appeared to be differentially expressed in pituitary tumor cells were identified. These bands were evaluated further by Northern blot analysis, using the PCR products as probes. Pituitary tumor-specific signals were detected for two bands. DNA sequencing analysis revealed that one sequence was homologous with insulin-induced growth response protein, while the other showed no homology to known sequences in the GenBank at the time of isolation. This 396-bp fragment (Fig. 1Go, a and b) detected a highly expressed mRNA of about 1.3 kb in pituitary tumor cells, but not in normal pituitary or in osteogenic sarcoma cells (Fig. 1cGo).



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Figure 1. Identification of PTTG by RNA Differential Display PCR

A, PCR of RNA from pituitary tumor cells and normal pituitary using 5' AAGCTTTTTTTTTTTG 3' as the anchored primer and 5' AAGCTTGCTGCTC 3' as an arbitrary primer. The differentially displayed band is indicated by an arrow. B, PCR reamplification of the differentially displayed band in panel A. C, Northern blot analysis using reamplified PCR product in panel B as the probe. GC and GH4, GH- and PRL-secreting pituitary tumor cells, respectively; Pit, normal pituitary; UM108, ostegenic sarcoma cells. Equal loading of RNA is indicated by 28S and 18S ribosomal bands.

 
To characterize this pituitary tumor-specific mRNA further, a cDNA library was constructed using mRNA isolated from rat pituitary tumor cells. Using the 396-bp PCR fragment as a probe, a cDNA clone of 974 bp was isolated and characterized. This cDNA was designated as PTTG. The sequence of PTTG contains an open reading frame for 199 amino acids (Fig. 2Go). The presence of an in-frame stop codon upstream of the predicted initiation codon indicates that PTTG contains the complete open reading frame. This was verified by demonstrating both in vitro transcription and in vitro translation of the gene product. As shown in Fig. 3Go, translation of in vitro transcribed PTTG sense mRNA results in a protein of approximately 25 kDa on SDS-PAGE, whereas no protein was generated in either the reaction without added mRNA or when PTTG antisense mRNA was used.



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Figure 2. DNA and Amino Acid Sequence of PTTG

The translation initiation codon is underlined and italicized, and the stop codons are underlined.

 


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Figure 3. In vitro Transcription and Translation of the PTTG

In vitro transcribed and translated PTTG product was analyzed by SDS-PAGE. The PTTG protein is indicated by an arrow; mol wt marker is indicated.

 
A subsequent data base search using BLAST EST program showed some homology between the coding sequence of PTTG and several partial transcripts of unknown function expressed during mouse embryonic development and in ovarian cancer. The highest homology was to a mouse embryonic cDNA sequence (mi66d08. rl). Protein profile analysis (BLAST Program search of databases of the National Center for Biotechnology Information), however, indicated that PTTG shares no homology with known protein sequences, and it is highly hydrophilic and contains no well recognized functional motifs.

Tissue Distribution of PTTG
The tissue expression pattern of PTTG mRNA was studied by Northern blot analysis. Figure 4AGo shows that among adult tissues examined, testis is the only tissue that expresses PTTG mRNA, and the testis expression level is much lower (2 mg polyA+ mRNA, 24 h exposure) than in pituitary tumor cells (20 mg total RNA, 6 h exposure, Fig. 2Go). PTTG is also expressed at low levels in embryonic liver (Fig. 4BGo). Hybridization to ß-actin control probe revealed appropriate transcripts for all RNA samples. Interestingly, the transcript in both testis and fetal liver (~ 1 kb) is shorter than the transcript in pituitary tumors (1.3 kb), suggesting that the mRNA may be either differentially spliced or uses alternate promoters or polyadenylation sites in these tissues, and that the 1.3-kb transcript is specific for pituitary tumor cells.



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Figure 4. PTTG mRNA Expression in Normal Tissues

Panel A, PolyA+ RNA from eight different rat tissues (indicated at the top of the figure) were used for Northern blot analysis. Top panel shows hybridization to PTTG probe (indicated by an arrow). Bottom panel shows hybridization to the control ß-actin probe. Molecular weight marker is shown on the side. Exposure time was 24 h for the PTTG probe and 2 h for the actin probe. Panel B, PolyA+ RNA from four different human fetal tissues (indicated at the top of the figure) were used for Northern blot analysis. Hybridization to PTTG and ß-actin probes are indicated by arrows.

 
Overexpression of PTTG in NIH 3T3 Cells Inhibits Cell Proliferation
It is difficult to predict the function of PTTG because the predicted protein sequence of PTTG does not contain recognizable motifs. Because PTTG mRNA is overexpressed in pituitary tumor cells, we initially sought to determine whether this protein exerts an effect on cell proliferation and transformation. Thus, an eukaryotic expression vector containing the entire coding region of PTTG was stably transfected into NIH 3T3 fibroblasts. Expression levels of the PTTG were monitored by immunoblot analysis using a specific polyclonal antibody directed against the first 17 amino acids of the protein. Expression levels of individual clones varied (Fig. 5Go), and clones that expressed higher protein levels were used for further analysis (Fig. 5Go, lanes 3, 4, 8, 9, and 10). NIH 3T3 cells seem to express low level endogenous PTSG protein, as a faint band is detectable by anti-PTTG antibody (Fig. 5Go, lane C).



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Figure 5. Overexpression of PTTG in NIH 3T3 Fibroblast Cells: Western Blot Analysis

The specific band detected by an anti-PTTG polyclonal antibody is indicated by an arrow. C, 3T3 cells transfected with pCMV vector without the insert; lanes 3–10 indicate individual clones transfected with PTTG expression vector. Molecular weight markers are indicated.

 
A nonradioactive cell proliferation assay (22, 23) was used to determine the effect of PTTG protein overexpression on cell proliferation. Figure 6Go shows that growth rate of 3T3 cells expressing PTTG protein (assayed by cellular conversion of tetrazolium into formazan) was suppressed 25 to 50% as compared with 3T3 cells expressing the pCMV vector alone, indicating that PTTG protein inhibits cell proliferation. The suppression of cell proliferation by PTTG protein is not surprising in view of the fact that pituitary adenomas are invariably slow-growing both in rat (24) and in humans (3).



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Figure 6. Effect of PTTG Expression on Cell Proliferation

Cell growth rate is expressed as absorbance at 595 nm. Error bars represent SEM (n = 6). Three independent experiments were performed.

 
PTTG Induces NIH 3T3 Cell Transformation and Generates Tumors in Nude Mice
The transforming property of PTTG protein was demonstrated by its ability to form foci in monolayer cultures and show anchorage-independent growth in soft agar (Fig. 7Go and Table 1Go). As primary pituitary cells are an admixture of multiple cell types and they do not replicate in vitro, NIH 3T3 cells were employed. As shown in Fig. 7Go, NIH 3T3 parental cells and 3T3 cells transfected with pCMV vector do not form colonies on soft agar (panels A and B), whereas 3T3 cells transfected with PTTG form large colonies (panel C). In addition, focal transformation is observed in cells overexpressing PTTG protein (panel E), but cells expressing pCMV vector without the PTTG insert showed similar morphology to the parental 3T3 cells (panel D). PTTG significantly induced the efficiency of colony formation up to 1.32% as compared with 0.013% for vector-only transfectants (Table 1Go).



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Figure 7. PTTG Induces Morphological Transformation and the Soft-Agar Growth of NIH 3T3 Cells

Cells (104 per dish) were plated as described in Materials and Methods. a, b, and c, Colony growth of the parental, pCMV-transfected, and pCMV-PTTG-transfected NIH 3T3 cells, respectively. d and e, Morphology of the pCMV vector alone and pCMV-PTTG-transfected NIH 3T3 cells, respectively.

 

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Table 1. Colony Formation by NIH 3T3 Cells Transfected with PTTG cDNA Constructs

 
To determine whether PTTG is tumorigenic in vivo, PTTG-transfected 3T3 cells were injected subcutaneously into athymic nude mice. All injected animals developed large tumors (1–3 g) within 3 weeks (Fig. 8Go and Table 2Go). No mouse injected with vector-only transfected cells developed tumors. Thus, PTTG is a potent transforming gene in vivo.



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Figure 8. PTTG Induces Tumor Formation in Nude Mice

Cells (3 x 105) were injected subcutaneously into athymic nude mice. Photograph was taken 3 weeks after injection. PTTG, Mouse injected with 3T3 cells transfected with PTTG expression vector; C, mouse injected with 3T3 cells transfected with pCMV vector alone.

 

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Table 2. In Vivo Tumorigenesis by NIH 3T3 Cells Transfected with PTTG cDNA Expression Vector

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mechanisms for pituitary tumorigenesis involve a multistep cuscade of recently characterized molecular events (25). Although the importance of GSa mutant proteins in the development of GH-secreting pituitary tumors is well established (4, 5), only about one third of these tumors contains these mutations, indicating the presence of additional transforming events in pituitary tumorigenesis. Despite efforts at screening for mutations of a variety of oncogenes and tumor suppressor genes in pituitary tumors, no other transforming gene has heretofore been identified.

In this study, we have taken a different approach to identify genes specifically expressed in pituitary tumor cells utilizing a recently developed RNA differential display assay (18, 19). We chose established GH- and PRL-secreting rat pituitary tumor cell lines to eliminate the admixture of normal tissues present in surgically excised human pituitary tumors or solid experimental rat tumors. Upon screening about 30% of expressed mRNA, a pituitary tumor-derived gene (PTTG) was identified and characterized. PTTG encodes for a protein of 199 amino acids that contains no characterized functional motif, suggesting that PTTG is a novel protein.

The pituitary tumor-specific expression of PTTG was shown by Northern blot analysis. Testis and fetal liver are the only normal tissues other than pituitary tumor cells that show PTTG expression. Interestingly, PTTG mRNA in testis and fetal liver is about 250 bp shorter than that of pituitary tumor, suggesting that it may represent a PTTG-splicing variant or alternative usage of promoters or polyadenylation sites. In view of the partial homology of PTTG to several mouse embryonic and ovarian cancer cDNAs, these observations suggest that PTTG may paly a role in fetal development and tumor formation in the ovary.

The importance of PTTG in tumorigenesis was illustrated by its ability to transform 3T3 fibroblasts when overexpressed in these cells, as shown by morphological change and anchorage-independent growth of PTTG transfectants in soft agar. Furthermore, nude mice injected with PTTG-expressing 3T3 cells developed large tumors within 3 weeks at all injection sites. These data show that PTTG alone is capable of cellular transformation, without the requirement of a complimentary oncogene, and that it is potently tumorigenic in vivo. Generally, full-cell transformation requires two complementary oncogenes (26, 27, 28). However, overexpression of a single oncogene may be sufficient to induce cellular transformation as shown in Rat-1 cell transformation by overexpression of Ras alone (29). Interestingly, PTTG does not stimulate but rather inhibits cell proliferation (within 72 h of assaying time) in cultured cells. This antiproliferative effect is similar to that seen with transforming growth factor-ß, which exerts potent inhibition of cell growth (30). It is possible, however, that once cells are transformed, cell proliferation is accelerated, which results in rapid growth of tumors in nude mice. It is unlikely that PTTG represents an activated oncogene due to point mutations in its coding region, as the sequence of the normal testicular transcript is identical to the transcript of pituitary tumor cells in the coding region (L. Pei and S. Melmed, unpublished data). It is, however, possible that enhanced tissue expression of PTTG by an as yet unidentified mechanism, may contribute to its oncogenic function.

In summary, we have isolated a novel cDNA (PTTG) that is overexpressed in rat pituitary tumor cells, induces cellular transformation when overexpressed in NIH 3T3 fibroblasts, and is tumorigenic in nude mice, suggesting that PTTG may play a role in pituitary cell transformation and tumor formation. PTTG represents the first isolated transforming gene highly expressed in pituitary tumor cells. Its unique sequence and the presence of a shorter transcript in testis and fetal liver suggest that it may belong to a new family of transforming genes. Further characterization of PTTG and its related genes will provide more insights into molecular mechanisms for tumorigenesis in the anterior pituitary.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RNA Isolation, PCR Differential Display, and Northern Blot Analysis
GC and GH4 pituitary tumor cell lines and an osteogenic sarcoma cell line UM108 were grown in DMEM supplemented with 10% FBS. Normal Sprague-Dawley rat pituitaries were freshly excised. Total RNA was extracted from cultured cells and pituitary tissue using RNeasy kit (QIAGEN, Chatsworth, CA) according to manufacturer’s instructions. Trace DNA contamination in RNA preparations was removed by DNaseI (GenHunter Corp., Boston, MA) digestion. Complementary DNA was synthesized from 200 ng total RNA using MMLV reverse transcriptase (GenHunter) and one of three anchored primers (GenHunter). The cDNA generated was used in the PCR display. Three downstream anchored primers AAGCT11X (where X may be A, G, or C), were used in conjunction with 40 upstream arbitrary primers for PCR display. We used 120 primer pairs to screen mRNA expression in pituitary tumors vs. normal pituitary. One tenth of the cDNA generated from the RT reaction was amplified using AmpliTaq DNA polymerase (Perkin Elmer, Norwalk, CT) in a total volume of 20 µl containing 10 mM Tris, pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 2 µM deoxynucleoside triphosphates, 0.2 µM each primer and 1 µl [35S]dATP. PCR cycles consisted of 30 sec at 94 C, 2 min at 40 C, and 30 sec at 72 C for 40 cycles. The products were separated on 6% sequencing gels, and dried gels were exposed to Kodak film for 24–48 h. After development, The DNA fragments from pituitary tumor and normal pituitary were compared. Bands unique to pituitary tumor were excised from the gel, and DNA was extracted by boiling in 100 ml water and precipitated with ethanol in the presence of glycogen (GenHunter). DNA was reamplified using the original set of primers and the same thermal cycling conditions except that the deoxynucleoside triphosphate concentration was increased to 20 mM. Reaction products were run on 1% agarose gel and stained with ethidium bromide. Bands were excised from the gel, eluted (QIAGEN), cloned in to TA vectors (Invitrogen, San Diego, CA) and sequenced using sequenase (USB, Cleveland, OH). For Northern blot analysis, 20 µg total RNA were fractionated on 1% agarose gel, blotted on to nylon membrane, and hybridized with random primed probe using Quickhyb solutions (Stratagene, La Jolla, CA). After washing, membranes were exposed to Kodak films for 6–72 h. A rat multiple tissue and a human fetal tissue Northern blot were purchased from Clontech. Approximately 2 µg poly A+ RNA per lane from eight different rat tissues were run on a denaturing formaldehyde 1.2% agarose gel, transferred to nylon membrane, and UV-cross linked. The membrane was first hybridized to the full-length PTTG cDNA probe and was stripped and rehybridized to a human ß-actin cDNA control probe. Hybridization was performed at 60 C for 1 h in ExpressHyb hybridization solution (Clontech). Washing was twice for 15 min at room temperature in 2x NaCl-sodium citrate, 0.05% SDS, and twice for 15 min at 50 C in 0.1% NaCl-sodium citrate, 0.1% SDS.

cDNA Library Construction, Screening, and DNA Sequencing
Poly A+ RNA was isolated from pituitary tumor GH4 cells using mRNA isolation kit (Stratagene, La Jolla, CA) according to manufacturer’s instructions, and was used to construct a cDNA library in ZAP Express vectors (Stratagene). The cDNA library was constructed using ZAP Express cDNA synthesis and Gigapack III gold cloning kit (Stratagene) after manufacturer’s instructions. The library was screened using the 396-bp differentially displayed PCR product (cloned into TA vector) as the probe. After tertiary screening, positive clones were excised by in vivo excision using helper phage. The resulting pBK-CMV phagemid containing the insert was identified by Southern blotting analysis. Unidirectional nested deletions were made into the DNA insert using EXOIII/Mung bean nuclease deletion kit (Stratagene) after manufacturer’s instructions. Both strands of the insert DNA were sequenced using Sequenase (USB).

In Vitro Transcription and Translation
The sense and antisense PTTG mRNAs were in vitro transcribed using T3 and T7 RNA polymerase (Stratagene), respectively. The excess template was removed by DNase I digestion. The in vitro transcribed mRNA was translated in rabbit reticulocyte lysate (Stratagene). Reactions were carried out at 30 C for 60 min, in a total volume of 25 µl containing 3 µl in vitro transcribed RNA, 2 µl [35S]methionine (Dupont, Wilmington, DE) and 20 µl lysate. Translation products were analyzed by SDS-PAGE (15% resolving gel and 5% stacking gel), and exposed to Kodak film for 16 h.

Overexpression of PTTG in NIH 3T3 Cells and Western Blot Analysis
The entire coding region of the PTTG was cloned in frame into pBK-CMV eukaryotic expression vector (Stratagene) and transfected into NIH 3T3 cells by calcium precipitation. Forty eight hours after transfection, cells were diluted 1:10 and grown in selection medium containing 1 mg/ml G418 for 2 weeks when individual colonies were isolated. Cell extracts were prepared from each colony, separated on 15% SDS-polyacrylamide gels, and blotted onto nylon membrane. A polyclonal antibody was generated using the first 17 amino acids of PTTG as epitope (Research Genetics, Huntsville, AL). The antibody was diluted 1:5000 and incubated with the above membrane at room temperature for 1 h. After washing, the membrane was incubated with horseradish peroxidase-labeled secondary antibody for 1 h at room temperature. The hybridization signal was detected by enhanced chemiluminescence (ECL detection system, Amersham, Arlington, IL).

Cell Proliferation Assay
Cell proliferation was assayed using CellTiter 96 nonradioactive cell proliferation assay kit (Promega, Madison, WI) according to the manufacturer’s instructions. Five thousand cells were seeded in 96-well plates (six wells for each clone in each assay), and incubated at 37 C for 24–72 h. At each time point, 15 µl of the dye solution were added to each well and incubated at 37 C for 4 h. One hundred microliters of the solubilization/stop solution were then added to each well. After 1 h of incubation, the contents of the wells were mixed, and absorbance at 595 nm was recorded using an ELISA reader. Absorbance at 595 nm correlates directly with the number of cells in each well.

PTTG Transformation in Vitro and in Vivo
For soft agar assay (31), 60-mm tissue culture plates were coated with 5 ml soft agar (20% 2x DMEM, 50% DMEM, 10% FBS, 20% 2.5% agar, melted and combined at 45 C). Two milliters of cells suspended in medium were then combined with 4 ml agar mixture, and 1.5 ml of this mixture were added to each plate. Cells were plated at a density of 104 cells per dish and incubated for 14 days before counting the number of colonies and photography. Cells (3 x 105) of either PTTG or pCMV vector alone transfected cells were resuspended in PBS and injected subcutaneously into nude mice (five animals for each group). Tumors were excised from animals at the end of the third week and weighed.


    FOOTNOTES
 
1 Address requests for reprints to: Shlomo Melmend, Division of Endocrinology and Metabolism, Cedars-Sinai Research Institute-UCLA School of Medicine, 8700 Beverly Boulevard, B131 Los Angeles, California 90048. Back

Supported by NIH Grants DK-42742 (S.M.) and DK-02346 (L.P.) and the Doris Factor Molecular Endocrinology Laboratory.

2 The GenBank accession number for PTTG is: U73030. Back

Received for publication October 28, 1996. Revision received January 17, 1997. Accepted for publication January 21, 1997.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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