Human Pituitary Tumor-Transforming Gene (PTTG1) Motif Suppresses Prolactin Expression

Gregory A. Horwitz, Irina Miklovsky, Anthony P. Heaney, Song-Guang Ren and Shlomo Melmed

Cedars-Sinai Research Institute-University of California Los Angeles School of Medicine, Los Angeles, California 90048

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary tumor-transforming gene (PTTG) originally isolated from GH-secreting pituitary adenoma cells causes in vitro cell transformation, in vivo tumorigenesis, and induces basic fibroblast growth factor. These functions require an intact C-terminal proline-proline-serine-proline motif. PTTG1 is abundantly expressed in human pituitary tumors and plays a role in the early stages of experimental prolactinoma formation. We now determined direct effects of PTTG1 on hormonal phenotypes of functional pituitary tumor cells. Overexpression of PTTG1 C terminus (amino acids 147–202) containing intact proline-proline-serine-proline motifs in rat prolactin (PRL)- and GH-secreting GH3 cells markedly abrogates PRL mRNA expression by more than 90% (P < 0.001) and hormone levels (P < 0.001) and PRL promoter activity (P < 0.01) compared with control vector cells or to a PTTG1 C terminus mutant (P163A, S165Q, P166L, P170L, P172A, and P173L). Wild-type PTTG1 C-terminal transfectants formed smaller (P < 0.05) sc tumors in rats compared with control or mutated PTTG1 C-terminal transfectants. Estrogen (10 nM) treatment for 48 h partially restored PRL expression in stable wild-type PTTG1 C-terminal transfectants. These results indicate that targeting PTTG1-mediated signaling alters the hormonal phenotype in pituitary cells and disrupted PTTG1 action may be a potential subcellular therapeutic tool for repressing PRL hypersecretion.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PITUITARY TUMOR-TRANSFORMING gene (PTTG1) was first isolated from rat GH-secreting cells by differential RNA display (1). The human PTTG homolog (PTTG1) is a member of a gene family (2), and like its rat counterpart, human PTTG1 causes in vitro cell transformation and in vivo tumor induction (3). We and others (4, 5, 6) previously demonstrated abundant PTTG1 expression in human neoplasms including pituitary tumors. PTTG1 overexpression induces basic fibroblast growth factor (bFGF) expression and secretion and PTTG is regulated by estrogen (2, 7). A proline-rich region in human PTTG1 contains two proline-proline-serine-proline (PXXP) motifs and are potential binding sites for signaling proteins containing an Src homology 3 (SH3) domain. Intact PXXP motifs are required for PTTG1 functions including transformation, bFGF induction, and angiogenesis (2, 8). PTTG1 is homologous to a Xenopus securin protein that maintains sister chromatid binding during mitosis (9). PTTG also transactivates DNA transcription (10); PTTG expression and intracellular localization are cell cycle-dependent and PTTG1 regulates endocrine tumor cell division and survival (11). Because PTTG1 is abundantly expressed in pituitary adenomas, we hypothesized that it plays a key role in pituitary tumorigenesis, and now determined direct effects of PTTG1 on hormonal phenotypes of pituitary tumor cells. The results show that overexpression of PTTG1 C-terminal peptide, containing intact PXXP motifs, in rat prolactin (rPRL)- and GH-secreting GH3 cells silences PRL gene expression. In contrast, no effect on PRL is observed in cells overexpressing dynamin containing an intact C-terminal PXXPSRP motif. Mutations at the PTTG1 PXXP motifs (P163A, S165Q, P166L, P170L, P172A, and P173L) inactivate PRL gene suppression, suggesting that targeted inhibition of PTTG1 action may be a potential subcellular tool for therapy of PRL-secreting adenomas.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Human PTTG1 C Terminus Overexpression Regulates PRL and GH Expression
To explore the impact of disrupted PTTG1 action on tumorigenesis, a truncated PTTG1 protein corresponding to amino acids 147–202 of the full-length coding sequence was introduced to interfere with intracellular signal transduction pathways. In another construct, the SH3-binding motif was mutated at P163A, S165Q, P166L, P170L, P172A, and P173L. Mutant and wild-type human PTTG1 C terminus cDNAs were cloned into the pCI-neo mammalian expression vector under control of the cytomegalovirus promoter, stably transfected into GH3 cells, and confirmed by Northern analysis (Fig. 1Go). RT-PCR followed by direct sequence analysis confirmed expression of wild-type and mutant PTTG1 C terminus in transfectants (data not shown). GH3 cells overexpressing wild-type PTTG1 C terminus exhibited approximately 10-fold decreased PRL secretion (V = 129.40 ng/ml, wild-type PTTG1 C terminus = 22.08 ng/ml, and mutant PTTG1 C terminus 306.88 ng/ml) and approximately 4-fold increased GH secretion (V = 35.31 ng/ml, wild-type PTTG1 C terminus = 131.31 ng/ml, and mutant PTTG1 C terminus = 30.31 ng/ml; Fig. 2Go, A and B). Three independent clones from each group (vector, wild-type PTTG1 C terminus, and mutant PTTG1 C terminus) were analyzed.



View larger version (40K):
[in this window]
[in a new window]
 
Figure 1. Wild-Type and Mutant PTTG1 C-Terminal Expression in Transfected GH3 Cells

A representative Northern blot in which 10 µg total RNA derived from each stable clone were hybridized with human PTTG1 C-terminal cDNA probe (top panel) or ß-actin (bottom panel). Vector, Cell line transfected with vectors only. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Mutant C-term, Cell line transfected with mutant PTTG1 C terminus expression vector.

 


View larger version (30K):
[in this window]
[in a new window]
 
Figure 2. PRL and GH Expression in Wild-Type and Mutant PTTG1 C-Terminal Transfectants

Transfectants were grown for 72 h in serum-containing medium when (A) PRL and (B) GH concentrations were measured in the medium. A representative Northern blot was performed in which 10 µg total RNA derived from each stable clone were hybridized with (A) rPRL cDNA probe or (B) rat GH cDNA probe. The bottom blot represents ß-actin expression. Vector, Cell line transfected with vectors only. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Mutant C-term, Cell line transfected with mutant PTTG1 C terminus expression vector. Each bar represents mean ± SEM of three wells, from a representative experiment repeated three times with similar results. *, P < 0.001.

 
Cells overexpressing wild-type PTTG1 C terminus exhibited approximately 13-fold decreased PRL mRNA expression (V = 1.5, wild-type PTTG1 C terminus = 0.13, and mutant PTTG1 C terminus = 1.9) and approximately 4-fold increased GH mRNA expression (V = 0.27, wild-type PTTG1 C terminus = 1.7, and mutant PTTG1 C terminus = 0.61) in comparison to cells overexpressing control vector alone or mutant PTTG C terminus as assayed by densitometry (Fig. 2Go, A and B). To test direct effects of wild-type and mutant PTTG1 C terminus on PRL gene transcription, the rPRL-luciferase reporter construct was transiently transfected into different stable transfectants and maintained in F10 medium containing 15% horse serum, 2.5% fetal bovine serum, and penicillin/streptomycin for 48 h. Cells overexpressing wild-type PTTG1 C terminus exhibited approximately 10-fold decreased PRL promoter-driven luciferase activity expression compared with control or mutant transfectants (V = 17.77 average fold increase, wild-type PTTG1 C terminus = 1.50 average fold increase, and mutant PTTG1 C terminus = 11.05 average fold increase; Fig. 3Go).



View larger version (18K):
[in this window]
[in a new window]
 
Figure 3. Effects of Wild-Type PTTG1 C-Terminal Peptide on PRL Transcription in GH3 Cells

Cells were transiently transfected with pA3.luc or pA3–425rPRL.luc reporter construct, and harvested 48 h later. Promoter activity is expressed as light emission units integrated over 15 sec. Vector, Cell line transfected with vectors only. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Mutant C-term, Cell line transfected with mutant PTTG1 C terminus expression vector. Each bar represents mean ± SEM of three wells per sample from a representative experiment repeated three times with similar results. *, P < 0.001.

 
As treatment of GH3 cells with bFGF induces PRL expression and secretion (12), transfectants were treated with 1 ng/ml bFGF for 24 h. As expected, GH3 parental, control vector, and mutant PTTG1 C terminus overexpressing cells all increased PRL mRNA and hormone levels. However, bFGF failed to induce PRL mRNA or hormone levels in cells overexpressing wild-type PTTG1 C terminus (Fig. 4Go).



View larger version (84K):
[in this window]
[in a new window]
 
Figure 4. Effects of bFGF Treatment on PRL Expression in GH3 Stable Transfectants

GH3 transfectants were incubated overnight, serum starved, and then treated with or without bFGF (1 ng/ml) for 24 h. A, Representative Northern blot in which 10 µg total RNA derived from each stable clone hybridized with rPRL cDNA probe (top panel) or ß-actin (bottom panel). B, PRL secretion in conditioned medium was measured. (-), Vehicle and (+), bFGF treated. Vector, Cell line transfected with vectors only. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Mutant C-term, Cell line transfected with mutant PTTG1 C terminus expression vector. Each bar represents mean ± SEM of a total of four wells derived from two separate experiments.

 
As estrogen activates PRL expression and secretion (13), cells were cultured in medium supplemented with charcoal-stripped serum to deplete steroid concentrations and then replenished with 10 nM diethylstilbestrol for 48 h. As expected, GH3 parental, control vector, and mutant PTTG1 C terminus overexpressing cells all exhibited enhanced PRL mRNA and hormone levels by at least 16-fold. Interestingly, there was a partial recovery of PRL mRNA and hormone levels in cells overexpressing wild-type PTTG1 C terminus treated with estrogen. (Fig. 5Go). Control vector and mutant PTTG1 C terminus overexpressing cells exhibited approximately a 50- and 25-fold increase compared with untreated transfectants, respectively, whereas wild-type PTTG1 C terminus overexpressing cells exhibited approximately 11-fold PRL increase.



View larger version (83K):
[in this window]
[in a new window]
 
Figure 5. Effects of Estrogen Treatment on PRL Expression in GH3 Stable Transfectants

GH3 transfectants were incubated in charcoal-stripped serum medium and then treated with 10 nM of diethylstilbestrol for 48 h. A, Representative Northern blot in which 10 µg total RNA from each cell line treated with diethylstilbestrol were used to hybridize with rPRL cDNA probe (top panel) or ß-actin (bottom panel). B, Conditioned medium from cells treated with diethylstilbestrol were harvested, and PRL secretion was examined. (-), Vehicle and (+), diethylstilbestrol treated. Vector, Cell line transfected with vectors only. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Mutant C-term, Cell line transfected with mutant PTTG1 C terminus expression vector. Each bar represents mean ± SEM of a total of four wells from two separate experiments.

 
Rat Dynamin 2(aa) [Dyn 2(aa)] Overexpression Has No Effect on PRL Expression
To further confirm the specificity of human PTTG1 motif in regulating PRL expression, Dyn 2(aa), which contains a C-terminal proline-rich domain and binds an SH3 domain (PXXPSRP; Ref. 14) was overexpressed. Figure 6AGo depicts expression of the Dyn 2(aa)-EGFP carboxy-terminal fusion construct. A dynamin mutant, Dyn 2(aa), lacking the proline-rich domain [Dyn 2(aa) {Delta}PRD] was also overexpressed. Results show that PRL levels in GH3 cells overexpressing Dyn 2(aa) and Dyn 2(aa) {Delta}PRD are unchanged compared with parental GH3 cells (Fig. 6BGo).



View larger version (36K):
[in this window]
[in a new window]
 
Figure 6. Effects of Dynamin on PRL Expression in GH3 Stable Transfectants

A, A representative Western blot depicting the GFP-dynamin fusion constructs in GH3 stable transfectants. B, PRL concentrations measured in the medium. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Dyn 2, Full-length wild-type Dyn 2(aa). Dyn 2(aa) {Delta}PRD, Dyn 2(aa) lacking the proline-rich domain. Each bar represents mean ± SEM of a total of eight wells from two separate experiments. *, P < 0.001.

 
In Vivo Tumor Growth
To test the effects of PTTG C terminus on in vivo tumor growth, stable GH3 transfectants were injected sc into 4- to 5-wk-old female Wistar-Furth rats (two rats for each group). Rats injected with control vector, wild-type, and mutant C-terminal expressing cells all developed tumors by 9 wk after injection when animals were killed and tumor tissue excised and weighed. Tumors were homogenized and RNA extracted for Northern analysis to confirm ex vivo expression of wild-type and mutant PTTG1 C terminus in tumor tissue (Fig. 7Go). Tumors derived from transfected cells overexpressed either wild-type or mutant PTTG1 C terminus, respectively, whereas control tumors did not contain a transcript.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 7. Wild-Type and Mutant PTTG1 C-Terminal Expression in Excised Rat Tumor Tissue

A representative Northern blot in which 20 µg total RNA derived from each tumor was hybridized with human PTTG C-terminal cDNA (top panel) or ß-actin (bottom panel). Vector, Cell line transfected with vectors only. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Mutant C-term, Cell line transfected with mutant PTTG1 C terminus expression vector.

 
Similar to the in vitro results, circulating PRL levels were decreased (~16-fold, P < 0.01) and GH levels increased (~1.6-fold, P < 0.01) in rats injected with wild-type PTTG1 C terminus cells. PRL levels were expected to be higher in rats injected with mutant PTTG1 C terminus cells; however, this was not the case. This discrepancy cannot be explained; however, it can be suggested that multiple factors affect in vivo PRL expression and secretion that cannot be controlled. Northern analysis of excised tumor tissue revealed a modest decrease in PRL and increase in GH mRNA expression levels in tumor tissue of animals injected with wild-type PTTG C terminus (Fig. 8Go, A and B).



View larger version (37K):
[in this window]
[in a new window]
 
Figure 8. PRL and GH Secretion in Rats Injected with Wild-Type and Mutant PTTG1 C-Terminal Transfectants and in Excised Tumor Tissue

Vector, WT C-term, and mutant C-term transfectants (3 x 106 cells) were sc injected in rats and allowed to grow for 9 wk at which time blood was being collected for (A) PRL and (B) GH measurement. A representative Northern blot was performed in which 20 µg total RNA from each excised tumor was hybridized with (A) rPRL cDNA or (B) rGH cDNA or (C) ß-actin. Vector, Cell line transfected with vectors only. WT C-term, Cell line transfected with human PTTG1 C terminus expression vector. Mutant C-term, Cell line transfected with mutant PTTG1 C terminus expression vector.

 
Excised tumor tissue was weighed and tumors derived from transfectants overexpressing wild-type PTTG1 C terminus were smaller (0.30 ± 0.11 g, P < 0.05) as compared with rats injected with control vector (1.54 ± 0.84 g) or mutant PTTG1 C terminus transfectants (0.67 ± 0.27 g) up to 9 wk after injection (Fig. 9Go).



View larger version (56K):
[in this window]
[in a new window]
 
Figure 9. Tumor Growth by GH3 Transfectants in Female Wister-Furth Rats

Rats were injected sc with either 3 x 106 vector, wild-type (WT C-term), or mutant PTTG C terminus (mutant C-term) overexpressing cells. After 9 wk, rats were killed and tumors excised and weighed. Each bar represents n = 5 individual tumors. Bottom panel, A representative tumor from each group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Functional and nonfunctional pituitary adenomas are monoclonal (15), and mechanisms involved in their pathogenesis include: loss of tumor suppressor gene functions such as multiple endocrine neoplasia type-1, retinoblastoma, p53, and nm23; activating mutations of stimulatory G protein, activated cAMP-response element-binding proteins, and mutations in H-ras (16, 17). Several growth factors also play a role in pituitary tumorigenesis, including FGF-2, FGF-4 (hst), and TGF{alpha} (18).

PTTG was initially isolated from hormone-secreting rat pituitary tumor cells, and PTTG1 expression in normal tissue is minimal except for high levels detected in testis. Importantly, PTTG1 is abundantly expressed in several human neoplasms and in human pituitary adenomas (3, 4, 5, 6). PTTG1 increases bFGF expression and secretion, and its expression levels in pituitary adenomas correlates with bFGF expression (3, 7). As PTTG1 is also regulated by both bFGF and estrogen (7), disrupted PTTG1 action may reverse or decrease the growth of pituitary adenomas.

The truncated PTTG1 motif (amino acids 147–202) of the full-length PTTG1 cDNA contains two PXXP motifs, potential binding sites for intracellular signaling molecules containing SH3 domains (3, 18). Overexpression of this truncated construct in GH3 cells markedly abrogated PRL mRNA and hormone levels, with a concordant increase in GH mRNA and hormone levels. Mutations at PXXP inactivated the suppression of PRL indicating the importance of this region for PTTG function. Transient PRL promoter activity was also decreased in wild-type PTTG1 C-terminal transfectants.

Several hormones and growth factors stimulate transcription and secretion of PRL, including bFGF (12), and estrogen (13). Interestingly, bFGF failed to induce PRL, whereas estrogen partially restored PRL expression in transfectants overexpressing wild-type PTTG C terminus. As expected, PRL expression in control cells and transfectants was induced by bFGF and estrogen, suggesting that interference of PTTG1 signaling inhibits the action of these factors in active hormone-producing pituitary cells.

The growth potential of the stable transfectants was examined in vivo. After sc injection, all rats developed tumors, however, rats injected with wild-type PTTG1 C terminus had significantly smaller tumors compared with those injected with vector alone or mutant PTTG1 C-terminal transfectants. Circulating PRL and GH levels were concordant with the observed in vitro pattern in the wild-type PTTG1 C terminus transfectants; i.e. lower PRL and higher GH levels. This result also suggests that higher GH circulating levels do not necessarily reflect larger mammosomatroph tumors.

For several higher eukaryote genes, methylation may repress expression (19, 20, 21, 22). It has previously been shown that an alkylating agent induces the appearance of PRL-deficient GH3 variants at a high frequency (23). This repression of PRL expression could be reverted by the DNA methylation inhibitor 5-azacytidine (23), suggesting that the alkylating agent may promote specific methylation of the PRL gene itself, or a gene regulating its activity. As the PRL gene was present and apparently intact in the PTTG1 C-terminal transfectants, we treated cells with 5-azacytidine. PRL expression was not further induced by 5-azacytidine in control and mutant transfectants, but the drug restored PRL mRNA and hormone levels in wild-type PTTG1 C-terminal expressing cells (data not shown). GH expression was not induced in these cells by 5-azacytidine, indicating the specificity of drug action on the PRL gene. Using methylation-sensitive enzymes, Southern blot analysis showed that the PRL gene was intact with no changes in methylation status (data not shown), suggesting that specific methylation activity was not occurring on the PRL gene, but perhaps a gene regulating its activity. Interestingly, IGF-II gene imprinting also appears to be regulated by enhancer-related methylation (24), as does the loss of the Apaf-1 tumor apoptosis effector (25). Thus, the methylation status of a transactivating or enhancer-associated PRL regulator may determine PRL repression in these transfectants. This possibility requires more detailed investigation.

As histone deacetylation has been implicated in transcriptional repression (20, 22), we examined the possibility that PRL gene repression caused by wild-type PTTG1 C terminus may be sensitive to the histone deacetylase inhibitor trichostatin A. However, treatment of transfectants with trichostatin A did not restore PRL mRNA or hormone levels (data not shown). Thus, although 5-azacytidine restored PRL in these transfectants containing an apparently intact PRL gene, other epigenetic factors are likely involved in the observed gene repression.

The pituitary-specific transcription factor Pit-1 is critical for PRL gene expression (26). Pit-1 protein levels assessed by Western blot analysis was unchanged in these transfectants as compared with vector control or mutant PTTG1 C-terminal transfectants (data not shown). An alternatively spliced Pit-1 isoform represses PRL gene expression in transplanted GH3 cells (27), and this isoform was not detected in our transfectants (data not shown). Based on our previous observations that PTTG expression coincides with an early lactotrophic hyperplastic response, angiogenesis, and prolactinoma development (7, 8), PTTG may be acting on the PRL promoter. This is supported by the results showing that disrupted PTTG action decreased PRL promoter activity.

PTTG1 contains two possible SH3-binding sites characterized by two PXXP motifs located at the C terminus (3). Multiple SH3 domains have been identified and each shown to have a distinct binding preference (28). Overexpression of Dyn 2(aa), containing an SH3 binding domain (PXXPSRP), did not alter PRL levels, thus indicating that the observed effects of PTTG1 C terminus overexpression on GH3 cells are selective for PTTG action rather than the presence of SH3-binding sequences. PTTG1 increases bFGF production, PTTG1 is induced by both bFGF and estrogen (3, 7) and mutations of the PXXP motifs abrogate bFGF induction (3). The results shown here suggest that interference of endogenous PTTG1 action in GH3 cells abrogates bFGF and estrogen actions on PRL expression. Further unraveling of mechanisms for PTTG regulation of PRL expression may provide novel therapeutic approaches for reversing hyperprolactinemia.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
Rat pituitary tumor GH3 cells (producing GH and PRL), supplied by the American Type Culture Collection (Manassas, VA), were cultured in F10 medium containing 15% horse serum, 2.5% fetal bovine serum, and penicillin/streptomycin. For bFGF treatment, cells were serum starved overnight and treated with 1 ng/ml of bFGF for 24 h. For estrogen treatment, cells were maintained for 5 d in phenol red-free F10 medium with 15% horse serum and 2.5% fetal bovine serum that had been pretreated with dextran-coated charcoal medium before replenishment with 10 nM diethylstilbestrol (Sigma-Aldrich Corp., St. Louis, MO).

Site-Directed Mutagenesis and Construction of Expression Vectors for Wild-Type and Mutant Human PTTG1 C-Terminal Peptides
Point mutations on the proline-rich domain of wild-type PTTG1 peptide were generated by PCR-based site-directed mutagenesis. Two synthetic oligonucleotides, 5'-GATGCTCTCCGCACTCTGGGAATCCAATCTG-3' and 5'-TTCACAAGTTGAGGGGCGCCCAGCTGAAACAG-3', which would cause amino changes P163A, S165Q, P166L, P170L, P172A, and P173L in the wild-type PTTG1 protein, were used to amplify human PTTG1 cDNA cloned into pBlue-Script-SK vector (Stratagene, La Jolla, CA). Amplified mutated cDNA was then cloned into mammalian expression vector pCI-neo mammalian expression vector (Promega Corp., Madison, WI).

To generate wild-type and mutant PTTG1 C-terminal peptide expression vectors, the internal XbaI site of wild-type and mutant PTTG cDNA and the 3'-portion of these cDNAs were cloned into pCI-neo (Promega Corp.) via XbaI and NotI sites. In this clone, the ATG for M147 of full-length PTTG1 is used as an initiation codon, generating a peptide of 56 amino acid residues corresponding to 147–202 of full-length wild-type PTTG1.

Stable Transfection of Human PTTG1 C Terminus into GH3 Cells
Wild-type and mutant PTTG1 C terminus expression constructs (2.5 µg) were transfected into rat GH3 cells with Lipofectin (Life Technologies, Rockville, MD) according to the manufacturer’s protocol. Twenty-four hours after transfection, cells were serially diluted and selected with G418 (0.4 mg/ml) for 4 wk. Individual clones were isolated and maintained in selection medium, and total RNA extracted from transfected cells. Expression of wild-type and mutated PTTG1 C terminus was confirmed by Northern blot analysis using the human PTTG1 C terminus fragment as a probe.

Stable Transfection of Rat Dynamin2 into GH3 Cells
Wild-type and mutant dynamin2 expression constructs (2.5 µg), cloned into the pEGFP vector, were transfected into rat GH3 cells using lipofectamine TM reagent according to manufacturer’s protocol. Twenty-four hours after transfection, cells were serially diluted and selected with G418 (1 mg/ml) for 3 wk. Resistant clones were isolated and maintained in selection medium. Expression of wild-type and mutant dynamin2 was confirmed by Western blot analysis using the monoclonal antibody to GFP (Roche Molecular Biochemicals, Indianapolis, IN).

Northern Blot Analysis
Total cell RNA was extracted from cell cultures (approximately 3 x 107 cells/group) and from excised rat sc tumors using TRIzol (Life Technologies, Inc.). Analysis was carried out as previously described (3).

Western Blot Analysis
GH3 transfectants were harvested from confluent 100-mm Petri dish with 500 µl of 2x sodium dodecyl sulfate sample loading buffer (100 mM Tris, pH 6.8; 4% sodium dodecyl sulfate; 0.2% bromophenol blue; 20% glycerol; and 200 mM ß-mercaptoethanol) and boiled for 5 min before loading. Protein expression in selected clones was confirmed by Western blot analysis using NuPage Bis-Tris Electrophoresis System (Invitrogen, Carlsbad, CA). Cellular proteins were separated on a 4–12% Bis-Tris gel under reducing conditions, and transferred to a polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA). Monoclonal antibody to GFP was used to detect GFP-dynamin fusion constructs.

Luciferase Assay
The reporter construct pA3.Luc containing only the luciferase reporter gene and pA3–425rPRL.luc (rPRL promoter) with the 498-bp fragment encompassing position -425 to +73 of the rPRL gene ligated upstream of the reporter gene in pA3.luc were used. A fusion gene containing ß-galactosidase gene under the control of the cytomegalovirus promoter was included as a control for transfection efficiency. Luciferase activity was assayed as previously described (29).

Hormone Assays
RIA for rat GH and PRL were performed in duplicate, using reagents provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD) as previously described (29).

In Vivo Tumor Growth
Transfectants (3 x 106 cells/group) expressing wild-type and mutant PTTG1 C terminus constructs and the original vector alone were injected sc into 4- to 5-wk-old female Wistar-Furth rats (Harlan Sprague Dawley, Inc.) as previously described (30). The use of rats was approved and followed guidelines outlined by the Institutional Animal Care and Use Committee.

Statistical Analysis
Results are expressed as mean ± SEM. Differences were assessed by one-way ANOVA. P < 0.05 was considered significant.


    ACKNOWLEDGMENTS
 
We thank Dr. Mark McNiven for providing the dynamin2 constructs.


    FOOTNOTES
 
This work was supported by NIH Grant CA-75979 and the Doris Factor Molecular Endocrinology Laboratory.

Abbreviations: bFGF, Basic fibroblast growth factor; Dyn 2(aa), rat dynamin 2(aa); PRD, proline-rich domain; PRL, prolactin; PTTG, pituitary tumor-transforming gene; PXXP, proline-proline-serine-proline; rPRL, rat PRL; SH3, Src homology 3.

Received for publication April 6, 2001. Accepted for publication January 8, 2003.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Pei L, Melmed S 1997 Isolation and characterization of a pituitary tumor-transforming gene (PTTG). Mol Endocrinol 11:433–441[Abstract/Free Full Text]
  2. Prezant TP, Kadioglu P, Melmed S 1999 An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family. J Clin Endocrinol Metab 84:1149–1152[Abstract/Free Full Text]
  3. Zhang X, Horwitz GA, Prezant TR, Valentini A, Nakashima M, Bronstein MD, Melmed S 1999 Structure, expression, and function of human pituitary tumor-transforming gene (PTTG). Mol Endocrinol 13:156–166[Abstract/Free Full Text]
  4. Heaney AP, Singson R, McCabe CJ, Nelson V, Nakashima M, Melmed S 2000 Pituitary tumor transforming gene in colorectal tumors. Lancet 355:712–715[CrossRef][Medline]
  5. Zhang X, Horwitz GA, Heaney AP, Nakashima M, Prezant TR, Bronstein MD, Melmed S 1999 Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas. J Clin Endocrinol Metab 84:761–767[Abstract/Free Full Text]
  6. Dominguez A, Ramos-Morales F, Romero F, Rios RM, Dreyfus F, Tortolero M, Pinto-Toro JA 1998 hPTTG, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG. Oncogene 17:2187–2193[CrossRef][Medline]
  7. Heaney AP, Horwitz GA, Wang Z, Singson R, Melmed S 1999 Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis. Nat Med 5:1317–1321[CrossRef][Medline]
  8. Ishikawa H, Heaney AP, Yu R, Horwitz GA, Melmed S 2001 Human pituitary tumor-transforming gene induces angiogenesis. J Clin Endocrinol Metab 86:867–874[Abstract/Free Full Text]
  9. Zou H, McGarry TJ, Bernal T, Kirschner MW 1999 Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis. Science 285:418–422[Abstract/Free Full Text]
  10. Wang Z, Melmed S 2000 Pituitary tumor transforming gene (PTTG) transforming and transactivating activity. J Biol Chem 275:7459–7461[Abstract/Free Full Text]
  11. Yu R, Ren S-G, Horwitz GA, Wang Z, Melmed S 2000 Pituitary tumor transforming gene (PTTG) regulates placental JEG-3 cell division and survival: evidence from live cell imaging. Mol Endocrinol 14:1137–1146[Abstract/Free Full Text]
  12. Ferrara N, Schweigerer L, Neufeld G, Mitchell R, Gospodarowics D 1987 Pituitary follicular cells produce basic fibroblast growth factor. Proc Natl Acad Sci USA 84:5773–5777[Abstract]
  13. Maurer RA 1982 Estradiol regulates the transcription of the prolactin gene. J Biol Chem 257:2133–2136[Abstract/Free Full Text]
  14. McNiven MA, Kim L, Krueger EW, Orth JD, Cao H, Wong TW 2000 Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape J Cell Biol 151:187–198[Abstract/Free Full Text]
  15. Herman V, Fagin J, Gonsky R, Kalman K, Melmed S 1990 Clonal origin of pituitary adenomas. J Clin Endocrinol Metab 71:1427–1433[Abstract]
  16. Shimon I, Melmed S 1997 Pituitary tumor pathogenesis. J Clin Endocrinol Metab 82:1675–1681[Free Full Text]
  17. Heaney AP, Melmed S 2000 New pituitary oncogenes. Endocr Relat Cancer 7:3–15[Abstract/Free Full Text]
  18. Yu H, Chen JK, Feng S, Dalgarno DC, Brauer AW, Schreiber SL 1994 Structural basis for the binding of proline-rich peptides to SH3 domains. Cell 76:933–945[Medline]
  19. Newell-Price J, Clark AJL, King P 2000 DNA methylation and silencing of gene expression. Trends Endocrinol Metab 11:142–148[CrossRef][Medline]
  20. Antequera F, Bird A 1993 Developmental pattern of gene-specific DNA methylation in the mouse embryo and germ line. Genes Dev 6:705–714
  21. Boyes J, Bird A 1992 Targeted mutation of DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926[Medline]
  22. Robertson KD, Wolffe AP 2000 DNA methylation in health and disease. Nat Genet 1:11–19[CrossRef]
  23. Ivarie RD, Morris JA 1982 Induction of prolactin-deficient variants of GH3 rat pituitary tumor cells by ethyl methanesulfornate: reversion by 5-azacytidine, a DNA methylation inhibitor. Proc Natl Acad Sci USA 79:2967–2970[Abstract]
  24. Yun K, Jinno Y, Sohda T, Niikawa N, Ikeda T 1998 Promoter-specific insulin-like growth factor 2 gene imprinting in human fetal liver and hepatoblastoma. J Pathol 185:91–98[CrossRef][Medline]
  25. Soengas MS, Capodieci P, Polsky D, Mora J, Esteller M, Opitz-Araya X, McCombie R, Herman JG, Gerald W, Lazebnik YA, Cordon-Cardo C, Lowe SW 2001 Inactivation of the apoptosis effector Apaf-1 in malignant melanoma. Nature 409:207–211[CrossRef][Medline]
  26. Nelson C, Albert VR, Elsholtz HP, Lu LE-W, Rosenfeld MG 1988 Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor. Science 239:1400–1405[Medline]
  27. Day RN, Day KH 1994 An alternatively spliced form of Pit-1 represses prolactin gene expression. Mol Endocrinol 8:374–381[Abstract]
  28. Pawson T 1995 Protein modules and signaling networks. Nature 373:573–580[CrossRef][Medline]
  29. Shimon I, Huttner A, Said J, Spirina OM, Melmed S 1996 Heparin-binding secretory transforming gene (hst) facilitates rat lacotrope cell tumorigenesis and induces prolactin gene transcription. J Clin Invest 97:187–195[Abstract/Free Full Text]
  30. Yamashita S, Slanina S, Kado H, Melmed S 1986 Autoregulation of pituitary growth hormone messenger ribonucleic acid levels in rats bearing transplantable mammosomatotrophic pituitary tumors. Endocrinology 118:915–918[Abstract]