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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2004-2303
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3715-3723
Copyright © 2005 by The Endocrine Society

Expression and Functional Analysis of Pituitary Tumor Transforming Growth Factor-1 in Uterine Leiomyomas

Shaw-Jenq Tsai1, Shu-Jeg Lin1, Ya-Ming Cheng, Hsiu-Mei Chen and Lih-Yuh C. Wing

Departments of Physiology (S.-J.T., S.-J.L., L.-Y.C.W.) and Obstetrics and Gynecology (Y.-M.C.) and Institute of Basic Medicine (H.-M.C.), College of Medicine, National Cheng Kung University, Tainan 701, Taiwan

Address all correspondence and requests for reprints to: Dr. Lih-Yuh C. Wing, Department of Physiology, National Cheng Kung University Medical College, Tainan 701, Taiwan. E-mail: wing{at}mail.ncku.edu.tw.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pituitary tumor-transforming gene-1 (PTTG-1) is a novel protooncogene overexpressed in numerous cancer cell lines and cancers. In this study we elucidate the expression of PTTG-1 in uterine leiomyomas and its functional role in the development of this disease. By comparing 23 pairs of leiomyomas and matched pairs of myometria, we found that the expression of PTTG-1 is significantly elevated in leiomyoma. The expression of PTTG-1 is independent of the menstrual cycle and is not affected by ovarian hormones. In contrast, basic fibroblast growth factor (bFGF) time- and dose-dependently stimulates PTTG-1 expression, which results in increasing cell proliferation. Forced expression of PTTG-1 by transient transfection stimulates bFGF and VEGF expression as well as changes the expression pattern of cell cycle proteins. Western blotting analysis demonstrates that the expressions of PTTG-1, bFGF, and the cell proliferation marker, proliferating cell nuclear antigen, are positively correlated with each other, which supports the hypothesis that the positive feedback loop between PTTG-1 and bFGF increases leiomyoma cell proliferation. In summary, we have shown for the first time that PTTG-1 is up-regulated in human uterine leiomyomas and that the positive feedback loop between PTTG-1 and bFGF may be pivotal in the growth of leiomyoma cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
UTERINE LEIOMYOMAS, OR fibroids, are typically defined as benign tumors of the myometrial smooth muscle tissue containing an abundant extracellular matrix. It is the most common gynecological neoplasm, with a prevalence rate as high as 30% in women older than 30 yr (1). Leiomyomas are monoclonal tumors derived from a single myometrial cell (2). Most leiomyomas are asymptomatic, but up to 25% of women older than 30 yr have significant symptoms, such as abnormal uterine bleeding, pelvic pain, and reproductive dysfunction (3). The incidence and severity of the symptoms associated with leiomyomas vary with their size, number, and location. In most countries, uterine leiomyomas are the most frequent indication for hysterectomy in premenopausal women and thus represent a major public health issue (1).

An abundance of evidence supports the hypothesis that the growth of leiomyomas is dependent on ovarian hormone-mediated, locally produced peptide growth factors. Progesterone and 17ß-estradiol (E2) cooperatively stimulate leiomyoma cell proliferation by up-regulating epidermal growth factor and epidermal growth factor receptor, respectively (4). A number of studies have reported that levels of IGF-I and its receptor, basic fibroblast growth factor (bFGF), TGF-ß, and endothelin-1 receptor were more elevated in leiomyomas than in myometria (5, 6, 7, 8, 9). In addition, a recent study (10) demonstrated that estrogen down-regulates p53 protein content in cultured leiomyoma cells, indicating that deregulation of a cell cycle check point may be involved in the development of leiomyomas.

Pituitary tumor-transforming gene-1 (PTTG-1), or securin, is a novel protooncogene first identified in a rat pituitary tumor GH4 cell line (11). Overexpression of PTTG-1 induces NIH-3T3 cell transformation in vitro and generates tumors in nude mice in vivo (11). Subsequently, human PTTG-1 was cloned and was found to be highly expressed in all cancer cell lines and in many types of cancer (12, 13, 14, 15, 16, 17, 18), suggesting that it is a common and important factor for most types of malignant tumors.

Human (h) PTTG-1 is predominantly expressed in the cytoplasm, with partial nuclear localization. Translocation of hPTTG-1 from cytoplasm to nucleus is mediated by a PTTG-1-binding protein that possesses a nuclear localization signal at its C terminus (19). Currently, the known target genes of hPTTG-1 are bFGF, c-myc, and prolactin (15, 20, 21), although the cis-binding element of PTTG-1 remains uncharacterized. Nevertheless, overexpression of PTTG-1 usually results in increased cell cycle progression, induced transformation, and blockage of cell differentiation. PTTG-1 was recently identified as a mammalian securin that maintains the binding of sister chromatids during mitosis (22). The level of hPTTG-1 expression increases in rapidly proliferating cells and is regulated in a cycle-dependent manner, peaking in mitosis (23). At the end of the metaphase, PTTG-1 is degraded by an anaphase-promoting complex, thus removing the inhibition of separin, which, in turn, mediates the degradation of cohesin and the separation of sister chromatids (22, 24, 25, 26). Consistent with this function, overexpression of hPTTG-1 has been reported to cause aneuploidy (abnormal numbers of chromosome or chromosome segments) in MG-63 osteosarcoma cells (27).

The expression of PTTG-1 is highly associated with cell cycle regulation and cell proliferation. Because the most distinctive characteristic between a leiomyoma and normal myometrium is the continuous growth of leiomyoma cells, we sought to determine the expression pattern of PTTG-1 and the factors that regulate PTTG-1 expression in uterine leiomyomas. We also wanted to investigate the functional role of PTTG-1 in the regulation of growth of leiomyoma cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue collection

Uterine leiomyomas and paired healthy myometrial tissue (n = 23) were collected immediately from patients after hysterectomy or myomectomy at National Cheng Kung University Hospital. Half of the tissue was immersed in Hanks’ solution supplemented with HEPES and kept on ice for cell isolation. The other half was snap-frozen in liquid nitrogen and stored at –80 C for mRNA and protein isolation. All patients were premenopausal and were not receiving any estrogen-related hormone therapy. The hysterectomy specimens were sent for pathological diagnosis of uterine leiomyoma and dating of menstrual cycle. Samples were excluded if menstrual cycle dates could not be assigned. Eight samples were collected from the proliferative phase, and 15 samples were collected from the secretory phase of the menstrual cycle. Human ethics committee approval was obtained from the clinical research ethics committee at National Cheng Kung University Medical Center, and informed consent was obtained from each patient.

Cell purification and culture

Uterine leiomyoma and myometrial tissues dissected from endometrial cell layers were cut into small pieces and digested in 20 ml 0.2% type II collagenase at 37 C for 1 h. Leiomyoma and myometrial cells were collected using centrifugation at 500 x g for 10 min after removal of undigested tissue debris. Cell pellets were washed with 10 ml PBS twice, then seeded in plastic flasks (75 cm2; T75, Nunc, Roskilde, Denmark). All media used in the cell culture were prewarmed to 37 C. Cells were cultured in T75 flasks with 20 ml DMEM containing 10% fetal bovine serum (FBS) and antibiotics (100 µg/ml streptomycin and 100 U/ml penicillin G) in a humidified atmosphere of 5% CO2 and 95% air at 37 C. Medium was changed every 2 d until confluence was reached, then cells were subcultured. When subcultured cells reached 70% confluence, cultured medium was changed to serum-free, phenol red-free DMEM/Ham’s F-12 (Invitrogen Life Technologies, Inc., Carlsbad, CA) for 48 h after it had been washed twice with 1x PBS. After 48 h, medium was changed to fresh serum-free, phenol red-free DMEM/Ham’s F-12, and various treatments were applied.

RNA extraction and RT-PCR

Total RNA from leiomyoma and matched myometrial tissues were extracted using a kit (RNeasy Total RNA Kit, Qiagen, Valencia, CA) according to the manufacturer’s instructions. Total RNA from cell cultures was harvested using TRIzol reagent (Invitrogen Life Technologies, Inc.) according to the manufacturer’s instructions. The expressions of bFGF and vascular endothelial growth factor (VEGF) transcript were detected using simple RT-PCR. Total RNA (500 ng) was subjected to RT as previously described (28, 29). The RT reaction was performed at 42 C for 90 min and then at 95 C for 10 min to terminate the reaction. Five microliters of RT products were subjected to 30 cycles of PCR amplification using specific primers for human bFGF and VEGF (Table 1Go).


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  		TABLE 1. Primers list

 
Quantification of mRNA concentrations using standard curve quantitative competitive (QC)-RT-PCR methodology

We prepared native and competitive plasmids for in vitro transcription of native and competitive RNA as described previously (28, 30, 31). Specific primer pairs for hPTTG-1 and glyceraldehyde-3-phosphate dehydrogenase were designed according to sequences deposited in GenBank (Table 1Go). All plasmids were sequenced using an automated sequencer (ABI model 377, PerkinElmer, Boston, MA) for verification of the sequences. Each RNA aliquot was used only once to reduce variation due to potential degradation of RNA after repeated freezing and thawing. The detailed procedure for the QC-RT-PCR standard curve was described previously (29, 32, 33). In brief, after RT, a fixed amount of competitor and RT cDNA products were subjected to 30 cycles of amplification (30-sec denaturation at 95 C, 30-sec annealing at 55 C, and 30-sec elongation at 72 C), followed by final elongation at 72 C for 5 min. The PCR products were resolved on a 5% acrylamide gel, stained with ethidium bromide, and then placed on a UV illuminator equipped with a camera connected to a computer. The gel image was analyzed using AlphaImager software (Alpha Innotech Corp., San Leandro, CA).

Immunostaining and immunoblotting

Paraffin-embedded tissues were sectioned at 5-µm thickness, mounted onto polylysine-coated slides, deparaffinized, and rehydrated. The procedure for immunostaining was described previously (34). For Western blot analysis, leiomyoma and paired healthy myometrium tissues were homogenized on ice with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (5 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml aprotinin, 10 µg/ml pepstatin A, and 50 µg/ml leupeptin) and phosphatase inhibitors (2.5 mM sodium pyrophosphate and 1 mM Na3VO4). Homogenates were subsequently centrifuged, and the supernatants were collected. Treated cells were also harvested using RIPA buffer containing protease inhibitors. Samples were quickly heated to 95 C for 10 min with 6x sample dye (final concentration, 1x) and stocked at –80 C until used. An equal amount of protein (100 µg) was loaded into each lane, separated on a 12.5% SDS-PAGE gel, and transferred to a polyvinylidene difluoride membrane (PerkinElmer). The membrane was blocked in 5% nonfat milk at 4 C overnight, then incubated in first antibody (goat antihuman PTTG-1, 1:200; goat antihuman FGF2, 1:500; mouse antihuman proliferating cell nuclear antigen (PCNA), 1:1,000; rabbit antihuman cyclin D1, A, and B1, 1:1,000; or mouse antihuman ß-actin, 1:10,000) at 4 C overnight. After the membrane had been washed three times for 5 min each time with 0.05% 1x PBS with 0.05% Tween 20, it was additionally incubated with 5% nonfat milk containing horseradish peroxidase-conjugated secondary antibody (rabbit antigoat IgG, goat antirabbit IgG, or goat antimouse IgG; 1:5000) for 1 h at room temperature. After incubation, the membrane was washed with 0.05% 1x PBS with 0.05% Tween 20 for 1 h and detected using an enhanced chemiluminescence detection kit (PerkinElmer). The membrane was stripped with stripping buffer at 55 C for 30 min and was redetected using a second antibody as described above.

Construction of expression vector

To generate wild-type PTTG-1 expression vector, primers were designed (hPTTG-FLmRNA_F and hPTTG-FLmRNA_R) from a full-length PTTG-1 mRNA sequence (Table 1Go). Additional restriction sites, BamHI and XhoI, were added to the 5' termini of forward and reverse primers, respectively. Human total RNA (50 ng) was reverse transcribed as described above, heated at 95 C for 2 min, then subjected to 30 cycles of PCR amplification (30-sec denaturation at 95 C, 30-sec annealing at 51 C, and 30-sec elongation at 72 C), followed by final elongation at 72 C for 5 min. The PCR product was separated on a 0.7% agarose gel and visualized using an imaging system to check that 655 bp of the full-length PTTG-1 were amplified. The PCR product and vector (pCDNA 6.0 C, Invitrogen Life Technologies, Inc.) were digested by BamHI and XhoI at 37 C overnight, separated on 0.7% agarose, purified, and subjected to ligation. Positive clones were selected and confirmed by sequencing.

Transient transfection

Cells were plated in a 6-cm dish for 24 h and transfected using Lipofectamine methodology. For each transfection sample, 10 µg DNA and 20 µl Lipofectamine 2000 (Invitrogen Life Technologies, Inc, Grand Island, NY) were diluted in 0.5 ml Opti-MEM I medium (Invitrogen Life Technologies, Inc.) for 5 min, separately. Then both diluted DNA and Lipofectamine 2000 were mixed and incubated at room temperature for 20 min to allow the DNA-Lipofectamine complexes to form. After incubation, 1 ml of a mixture of antibiotic-free DMEM/Ham’s F-12 medium containing 10% FBS was added to the 6-cm dish. After 6 h, medium was replaced with fresh nonantibiotic medium containing 10% FBS. After incubation for 12, 24, and 48 h, cells were harvested using TRIzol reagent and a RIPA buffer for mRNA and protein determination, respectively.

[3H]Thymidine incorporation

Leiomyoma cells were seeded into a six-well plate (8 x 104/well) and cultured in serum-free, phenol red-free DMEM/Ham’s F-12 medium for 48 h. Medium was replaced with fresh serum-free, phenol red-free DMEM/Ham’s F-12 medium and treated with various concentrations of bFGF for another 48 h. During the final 6 h, cells were coincubated with 0.5 µCi [3H]thymidine. After incubation, cells were subjected to a [3H]thymidine incorporation assay as previously described (29, 35, 36). In brief, cells were washed twice using cold 1x PBS. After the addition of 10% trichloroacetic acid (1 ml/well) at 4 C for 20 min, trichloroacetic acid was removed by washing the cells twice with cold 1x PBS. The acid-insoluble fractions were dissolved by the addition of 1 N NaOH and were shaken at room temperature for 1 h. The contents were then neutralized with an equal volume of 1 N HCl to a final concentration of 0.5 N. Five hundred-microliter aliquots were transferred to a scintillation vial containing 3.5 ml scintillation cocktail (Ready Safe, Beckman Coulter, Inc., Fullerton, CA). The radioactivity was measured using a liquid scintillation counter.

Statistical analysis

Differences in PTTG-1 RNA expression in leiomyoma and myometrial tissues were analyzed using a paired t test. Correlation between levels of protein expression was performed using the Pearson rank-sum test. Differences between individual treatment groups and controls in PTTG-1 mRNA expression were analyzed using one-way ANOVA, followed by Dunnett’s test. Statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
PTTG-1 is overexpressed in uterine leiomyomas

To quantify the expression levels of human PTTG-1, we developed a standard curve QC-RT-PCR analysis specific for PTTG-1 (Fig. 1AGo). This enabled us to quantify the absolute amounts of RNA transcript with high accuracy and sensitivity. We found that the expression of PTTG-1 mRNA was significantly (P < 0.05) higher in leiomyomas than in matched pairs of myometria (Fig. 1BGo; n = 23). The levels of PTTG-1 mRNA from different sized leiomyomas in the same patient were not different (Fig. 1BGo and data not shown; n = 9); thus, the data were combined for analysis. Using Western blot analysis, the expression of PTTG-1 protein was higher in leiomyomas than in myometrial counterparts (Fig. 1CGo). Additional analysis revealed that expression of PTTG-1 was not dependent on menstrual cycle and was greater in leiomyomas in both the proliferative and secretory phases (Fig. 1DGo).



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  		FIG. 1. Expression of PTTG-1 mRNA and protein in paired myometrial and leiomyoma tissues. A, Construction of a standard curve for quantifying PTTG-1 mRNA. The upper panel shows a representative gel picture of ethidium bromide-stained PCR products amplified from native and competitor RNAs. A 2-fold serial dilution of native RNA (25.6 to 0.2 attomoles) was reverse transcribed and PCR amplified in the presence of 2 attomoles competitor. Lower panel, Standard curve produced from analyzing the intensity of bands shown in the upper panel. The band intensity was quantified by AlphaImager computer software to construct the standard curve. The inset shows two samples that were RT-PCR amplified in the presence of the same amount of competitor. The ratios of band intensity in lanes 1 and 2 were logarithmically transformed and compared with the standard curve to calculate the amount of transcript (vertical lines cross the x-axis). M, DNA molecular weight marker; NC, negative control omitting reverse transcriptase in the RT reaction. B, A representative picture of PTTG-1 transcripts amplified by RT-PCR in leiomyoma and myometrial tissues collected from the same individual (upper panel). Mean concentrations of PTTG-1 transcripts from leiomyoma (L; n = 32) and myometrium (M; n = 23) are shown in the lower panel. The expressions of PTTG-1 in different sized leiomyoma samples obtained from the subject (e.g. L2–1 and L2–2) were not different and were combined for analysis. *, Significant difference from myometrium (P < 0.05). C, A representative Western blot result showing three sets of PTTG-1 protein expressed in leiomyoma (L) and myometrial (M) tissues collected from the same individual. L1–1 and L1–2 denote two leiomyomas of different sizes collected from the same subject. D, The expression of PTTG-1 transcripts in leiomyoma and paired myometrium was divided into the proliferative phase (n = 8) and the secretory phase (n = 15) Data are expressed as the mean ± SEM. *, Significant difference from myometrium (P < 0.05). M, Myometrium; L, leiomyoma.

 
Overexpression of PTTG-1 is associated with cell proliferation

Immunohistochemical staining of the proliferative marker, PCNA, showed that more immunoreactive cells were identified in leiomyomas than in paired myometrium (Fig. 2AGo). Because immunostaining is not quantitative, we used Western blot to quantify the expression of PCNA in leiomyoma and myometrial tissues. Western blot analysis demonstrated that PCNA was elevated in leiomyomas (Fig. 2BGo). Correlation analysis revealed that the levels of PCNA and PTTG-1 in leiomyomas were positively associated (Fig. 2CGo; r = 0.590; P < 0.01).



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  		FIG. 2. Increased cell proliferation marker and PTTG-1 expression in leiomyoma tissues. A, A representative picture shows immunohistochemical staining of the proliferation marker, PCNA, in paired uterine myometrial (M) and leiomyoma (L) tissues. The myometrial tissue shows some cells that were weakly stained, whereas the leiomyoma tissue has much more strongly stained cells. This experiment was repeated five times using different pairs of samples, and results were similar. B, Western blot analysis of PCNA protein expression in paired leiomyoma and myometrial tissues. ß-Actin was used as an internal control. C, Correlation of levels of PTTG-1 and cell proliferation index using PCNA as a marker. Band intensities of PTTG-1 and PCNA Western blots were quantified using an AlphaImager system. The ratios of band intensities of leiomyoma to myometrium were calculated, expressed as protein induction, and used to plot against each other. Each dot represents an individual.

 
Effects of E2 and progesterone on expression of PTTG-1 in cultured leiomyoma cells

To determine whether the expression of PTTG-1 was regulated by ovarian hormones, leiomyoma cells were cultured without serum and treated with different doses of E2 and progesterone for 24, 48, 72, and 96 h. Treatment with E2 (1–100 nM; Fig. 3AGo and data not shown), progesterone (1–100 nM; Fig. 3BGo and data not shown), and a combination of E2 (1 nM) and progesterone (10 nM) failed to affect PTTG-1 expression (data not shown). In contrast, bFGF expression was induced by 1 nM E2 (Fig. 3CGo), indicating that cultured leiomyoma cells retained the ability to respond to ovarian steroids.



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  		FIG. 3. The expression of PTTG-1 transcripts in cultured leiomyoma cells is independent of E2 or progesterone (P4). Leiomyoma cells were cultured in serum-free, phenol red-free DMEM/Ham’s F-12 medium for 48 or 96 h in the presence or absence of different doses of E2 (A) or P4 (B). Levels of PTTG-1 mRNA were quantified by standard curve QC-RT-PCR. Data are expressed as the mean ± SEM from three experiments performed in triplicate using different batches of cells. C, Induction of bFGF in E2-treated leiomyoma cells. Cells were treated with vehicle (Con) or E2 (1 nM) for 96 h, and bFGF mRNA was determined by RT-PCR. Data are expressed as the mean ± SEM from three experiments using different batches of cells. *, Significant difference from vehicle-treated group at P < 0.05.

 
Effect of bFGF on expression of PTTG-1 in cultured leiomyoma cells

Because E2 and progesterone failed to induce PTTG-1 expression in cultured leiomyoma cells, we determined the effects of peptide growth factors on the expression of PTTG-1. bFGF (5 ng/ml) induced PTTG-1 expression (Fig. 4AGo). At 36 h after bFGF treatment, the expression of PTTG-1 was elevated, but did not reach statistical significance (P = 0.07). At 48 h, its expression was significantly increased (P < 0.05). A dose-effect experiment demonstrated that bFGF induced PTTG-1 expression in a dose-dependent manner (Fig. 4BGo). Western blot analysis showed that bFGF significantly (P < 0.05) induced PTTG-1 protein expression 48 h after treatment (Fig. 4CGo).



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  		FIG. 4. Effects of bFGF on PTTG-1 mRNA and protein expression. A, Human leiomyoma cells were treated with bFGF (5 ng/ml) for various times as indicated. Levels of PTTG-1 transcripts were measured by standard curve QC-RT PCR (n = 4 in triplicate). B, Leiomyoma cells (n = 5 in triplicate) were treated with bFGF at the concentration indicated for 48 h. Levels of PTTG-1 transcripts were measured by standard curve QC-RT PCR. C, Expression of PTTG-1 protein was induced by treatment with bFGF (5 ng/ml). The inset shows a representative Western blot picture. Data are expressed as the mean ± SEM from four experiments using different batches of cells. *, Significantly different from control group (P < 0.05).

 
Effect of bFGF on proliferation of leiomyoma cells

To determine whether bFGF-induced PTTG-1 expression would affect cell proliferation, the expression of the cell cycle regulators, cyclin D1, A, and B1, in bFGF-treated leiomyoma cells was examined. bFGF induced cyclin D1 expression as early as 12 h after treatment, and expression peaked at 24 h (Fig. 5AGo). Increased cyclin A and B1 expression was first evident 24 h after bFGF treatment and remained high thereafter (Fig. 5AGo). A [3H]thymidine incorporation assay showed that bFGF had also dose-dependently induced leiomyoma cell proliferation (Fig. 5BGo); the effective dose was as low as 0.5 ng/ml.



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  		FIG. 5. Induction of cyclin expression and leiomyoma cell proliferation by bFGF. A, Human leiomyoma cells were treated with bFGF (5 ng/ml) for various times as indicated. The expressions of cyclin D1, A, and B1 were detected by specific antibodies using Western blot analysis. This experiment was repeated three times, and similar results were obtained. B, Leiomyoma cells treated with various doses of bFGF in the absence of serum, and cell proliferation was analyzed by [3H]thymidine incorporation. Data are expressed as the mean ± SEM from three experiments performed in triplicate. *, Significant difference from the control at P < 0.05.

 
Effect of PTTG-1 overexpression on leiomyoma cells

Leiomyoma cells were transiently transfected with expression vector containing full-length human PTTG-1 cDNA, and the expressions of several gene products were quantified. The expression of exogenous PTTG-1 was observed by Western blot with an antihistamine tag antibody (Fig. 6AGo). Transient transfection of PTTG-1 resulted in decreased cyclin D1 and cyclin A protein expression, but increased cyclin B1 expression, 24 h after transfection (Fig. 6AGo). In addition, overexpression of PTTG-1 markedly induced bFGF and VEGF mRNA expression (Fig. 6BGo). This suggested that PTTG-1 can induce peptide growth factor production to facilitate cell growth and, more importantly, that a positive feedback loop between bFGF and PTTG-1 may exist in leiomyoma cells.



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  		FIG. 6. Functional roles of PTTG-1 in leiomyoma cells. A, Leiomyoma cells were transiently transfected with PTTG-1 expression vector and were harvested 24 h after transfection. Equal amounts of proteins were loaded, and specific antibody against histamine tag was used to detect exogenous PTTG-1 protein expression. The blot was then stripped and reprobed with different antibodies against cyclin A, B1, and D1 and ß-actin, sequentially. B, Leiomyoma cells were transiently transfected with PTTG-1, and levels of bFGF and VEGF transcripts were amplified by RT-PCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. These experiments were repeated three times using different batches of leiomyoma cells, and the results were similar.

 
Positive feedback loop between bFGF and PTTG-1 in uterine leiomyoma

Because bFGF significantly induced PTTG-1 expression, and overexpression of PTTG-1 led to increased bFGF expression, we reevaluated the expression pattern of bFGF in paired myometrial and leiomyoma samples. Western blot analysis showed that the expression of bFGF was also higher in leiomyoma than in healthy myometrium (Fig. 7AGo). Correlation analysis showed a positive correlation between bFGF and PTTG-1 in leiomyoma tissue (Fig. 7BGo; r = 0.517; P = 0.03).



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  		FIG. 7. Positive correlation between PTTG-1 and bFGF protein expression in leiomyoma tissues. A, A representative Western blot shows that expression of bFGF in paired myometrium (M) and leiomyoma (L) tissues. ß-Actin was used as an internal control. B, Western blot results of PTTG-1 (see Fig. 1CGo) and bFGF (A) from the same patient were quantified by AlphaImager system. The ratios of band intensities of leiomyoma to myometrium were calculated and expressed as protein induction. Each dot represents one pair of myometrium and leiomyoma samples. Six pairs of samples were dropped from this analysis, because bFGF was below the detection limit in myometrium, whereas the expression of bFGF in leiomyoma appeared to be normal (such as M1 in A).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have presented evidence demonstrating that PTTG-1, a cell cycle regulator and transcription factor, is overexpressed in human uterine leiomyoma cells compared with its expression in autologous myometrial cells. The expression of PTTG-1 is stimulated by bFGF, but not by E2 or progesterone, in cultured leiomyoma cells. In cultured leiomyoma cells, forced expression of PTTG-1 increases bFGF expression, which stimulates cell cycle progression. The positive feedback loop between PTTG-1 and bFGF augments both their expression and cell proliferation. Increased expression of PTTG-1 and bFGF in leiomyomas in vivo is positively correlated with the elevation of PCNA. This provides additional support for the hypothesis that overexpression of PTTG-1 is important in the development of leiomyomas.

Continuous growth of smooth muscle cells is a hallmark of uterine leiomyoma. The major determinants of leiomyoma growth are serum E2 and progesterone, which regulate the expression of several peptide growth factors or their cognate receptors (4, 37, 38, 39). These peptide growth factors have autocrine and paracrine actions to stimulate the proliferation of leiomyoma cells (8, 40, 41, 42, 43); however, how these factors cause cell growth remains unclear. Based on several studies, it has been observed that the effects of PTTG on cell proliferation are a function of the level of expression (14, 44). PTTG-1 is a transcription factor with ill defined DNA binding sites that can stimulate the expression of the protooncogene, c-myc. Overexpression of c-Myc protein results in increased cell proliferation, causing cell transformation and blocking differentiation (45). In addition, PTTG-1 per se is a critical factor that regulates cell cycle progression by holding sister chromatids together during the mitosis phase. PTTG-1 expression peaks during mitosis, and PTTG-1 degradation releases cells from the M phase to complete the cell cycle (23). We found that PTTG-1 was significantly higher in leiomyomas than in matched pairs of uterine myometria. Elevation of PTTG-1 is positively correlated with the cell proliferation marker in leiomyomas. Our current results agree with previous reports that more positively PCNA-immunoreacted cells are identified in leiomyomas than in myometrium (4) and extends our understanding that this is probably due to increased cell proliferation mediated by elevated PTTG-1.

That PTTG-1 expression in uterine myometrium and leiomyomas does not fluctuate during the menstrual cycle suggests that it is not dependent on ovarian sex hormones. We found that treatment with E2 or progesterone did not affect PTTG-1 expression in cultured leiomyoma cells. One report (46) suggests that it may be due to a lack of receptors in cultured leiomyoma cells. We think this is unlikely, because our RT-PCR data showed that ER{alpha} and progesterone receptor were both present in cultured primary leiomyoma cells (data not shown) and that bFGF mRNA was induced 96 h after E2 treatment in the same cells. Heaney et al. (47, 48) reported that E2 induced PTTG-1 expression in a rat pituitary tumor model. However, unlike rodent pttg-1, the human PTTG-1 gene lacks the cognate estrogen-responding element. Our results agree with a recent report (49) that E2 failed to induce PTTG-1 expression in a human glioma cell line. Thus, the function of E2 in human PTTG-1 expression may require additional investigation. To the best of our knowledge, there are no published reports about the association between progesterone and PTTG-1 expression. The failure of E2 and progesterone to induce PTTG-1 expression in the present study suggests that other factors cause increased PTTG-1 levels in uterine leiomyomas.

PTTG-1 and bFGF are coexpressed in several human cancers, including pituitary adenomas (47), nonsmall cell carcinomas (50), and thyroid cancer (51). Because PTTG-1 mRNA can be up-regulated by bFGF in NIH-3T3 cells (47), we tested whether bFGF could induce PTTG-1 expression in human uterine leiomyoma cells. We found that bFGF dose- and time-dependently increased PTTG-1 mRNA and protein levels in primary cultured leiomyoma cells, which agrees with the study of NIH-3T3 cells (47). The induction of PTTG-1 by bFGF in leiomyoma cells occurs along with increased DNA synthesis and cyclin A, D1, and B1 expression.

Overexpression of human PTTG-1 increased bFGF and VEGF mRNA expression. This indicates that PTTG-1 may be critically involved in the development of leiomyoma, because both bFGF and VEGF synergistically modulate angiogenesis, a rate-limiting step in the developmental pathway of solid tumors (52). Our data, showing decreases in cyclins A and D1 and an increase in cycle B1 24 h after transfection, are consistent with a report that overexpression of PTTG-1 resulted in an accumulation of cells at the G2 phase (53). The mechanism through which overexpression of PTTG-1 increases cells in the G2 and M phase is not clear. One possibility is that overexpression of PTTG-1 hastens cell cycle progression, which drives most cells to pass through the G1 and S phases relatively quickly. Alternatively, sustained elevation of PTTG-1 may prevent sister chromatid separation, thus impeding cells from exiting the M phase, much as the overexpression of PTTG-1 prolongs both prophase and metaphase in H1229 cancer cells (54). Finally, cell proliferation or arrest at the G2 and M phases is dependent on the levels of PTTG-1 protein; a low level induced cell proliferation and a high level inhibited mitosis (55); therefore, PTTG-1-induced cell cycle progression is still controversial and additional investigation is necessary. Nevertheless, that increased PTTG-1 expression results in increasing cell proliferation and tumor formation has been reported in numerous human and mouse models (12, 13, 14, 15, 16, 17, 18), which may support the causative role of PTTG-1 in leiomyoma growth.

The involvement of PTTG-1 in the transformation of cells and development of cancers is much more complex than previously known. The transforming ability and high level of expression of PTTG-1, but not its mutation in malignant tumors, have demonstrated that PTTG-1 is involved in tumorigenesis; however, the mechanisms used and the interaction of PTTG-1 with other oncogenes and tumor suppressor genes remain enigmatic. Recent studies also demonstrated the cooperative relationships between E2, bFGF, and PTTG-1 in endocrine-related cancers (17, 47, 48). In the present study we have reported evidence of a positive correlation among PTTG-1, bFGF, and proliferative marker in uterine leiomyoma and a positive feedback loop between PTTG-1 and bFGF in cultured leiomyoma cells. This autocrine/paracrine crosstalk may explain to some extent why antiestrogen or antiprogesterone treatments result in only a partial reduction in tumor size and fail to cause complete regression of leiomyoma, because the growth of leiomyoma cells is independent of ovarian steroids to some degree. Our current findings may shed light on developing new treatment regimens for leiomyoma by targeting the disruption of the local autocrine/paracrine feedback loop.


   Footnotes
 
This work was supported by Grants NSC93-2320-B-006-024 (to S.-J.T.) and NSC93-B-006-002 (to L.-Y.C.W.) from the National Science Council of Taiwan.

First Published Online March 15, 2005

1 S.-J.T. and S.-J.L. contributed equally to this study. Back

Abbreviations: bFGF, Basic fibroblast growth factor; E2, 17ß-estradiol; FBS, fetal bovine serum; h, human; PCNA, proliferating cell nuclear antigen; PTTG-1, pituitary tumor-transforming gene-1; RIPA, radioimmunoprecipitation assay; VEGF, vascular endothelial growth factor; QC-RT-PCR, quantitative competitive RT-PCR.

Received November 24, 2004.

Accepted March 4, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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