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Departments of Medicine (A.P.H., M.F., G.H.) and Pathology (V.N.), Cedars-Sinai Research Institute, University of California School of Medicine, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Anthony P. Heaney, M.D., Becker-126, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, B-2015, Los Angeles, California 90048. E-mail: heaneya{at}csmc.edu
Abstract
Thyroid tumors comprise a broad spectrum of neoplastic phenotypes, and distinct molecular events have been implicated in their pathogenesis. Pituitary tumor transforming gene, originally isolated from GH4 pituitary cells, is tumorigenic in vivo, regulates basic fibroblast growth factor secretion, and is homologous to a securin inhibitor of chromatid separation. Pituitary tumor transforming gene 1 is expressed at low levels in several normal human tissues and is abundantly expressed in neoplasms, including colorectal carcinoma, where pituitary tumor transforming gene expression correlated highly with tumor invasiveness. As pituitary tumor transforming gene is regulated by E and as thyroid cancer shows a strong female preponderance, we examined pituitary tumor transforming gene 1 expression and action in human thyroid tumors and in normal human and rat thyroid cells. Increased pituitary tumor transforming gene 1 expression was evident early in thyroid tumors and was most abundantly expressed in a subset of thyroid hyperplasia, follicular adenomas, and follicular carcinomas (1.8-fold; P < 0.0001). Pituitary tumor transforming gene 1 overexpression in rat FRTL5 thyroid cells and in primary human thyroid cell cultures causes in vitro transformation and produces a dedifferentiated neoplastic phenotype.
As pituitary tumor transforming gene 1 was abundantly overexpressed in follicular adenoma and follicular carcinoma, we propose that pituitary tumor transforming gene overexpression may play a role in the early molecular events leading to divergent development of follicular and papillary carcinoma.
THYROID CANCERS ACCOUNT for about 90% of newly diagnosed endocrine malignancies and for approximately 1200 deaths/yr in the U.S. These tumors comprise a broad spectrum of neoplastic phenotypes, including follicular adenomas, well differentiated follicular and papillary carcinoma, and invasive and almost universally fatal anaplastic carcinoma (1).
Distinct molecular events (2) occurring in thyroid
neoplasia include ras mutations, detected early in both
benign and malignant tumors (3, 4, 5), and activating TSH
receptor and Gs
-subunit mutations, reported in
some follicular carcinomas (6). 11q13 allelic losses are
found in follicular, but not papillary, lesions (7). In
contrast, ret rearrangements are common in
radiation-associated papillary carcinomas (8, 9, 10), and p53
inactivation marks the transition to aggressive follicular and the
anaplastic phenotypes (11).
Pituitary tumor transforming gene (PTTG), originally isolated from GH4 pituitary cells, is tumorigenic in vivo, regulates basic fibroblast growth factor (bFGF) secretion (12, 13), and is homologous to a securin inhibitor of chromatid separation (14). The human PTTG family consists of at least four homologous genes, of which PTTG1 is located on chromosome 5q33 (15) and is expressed at low levels in several normal human tissues and abundantly in neoplasms, including pituitary tumors (16, 17, 18). We recently demonstrated that pituitary pttg is regulated by E (19) and is also overexpressed in colorectal carcinoma (16).
As PTTG may predispose to chromosomal instability, and thyroid cancer shows a strongly female preponderance, we hypothesized that PTTG1 expression may be related to the divergent molecular events that occur in thyroid tumorigenesis.
We show here that early increased PTTG1 expression in thyroid tumors is most abundant in a subset of thyroid hyperplasia, follicular adenomas, and follicular carcinomas. We also demonstrate that PTTG1 overexpression in rat FRTL5 thyroid epithelial cells and primary human thyroid cell cultures causes in vitro transformation and produces a dedifferentiated phenotype. PTTG may therefore play a role in the early molecular events leading to divergent development of follicular and papillary carcinoma.
Subjects and Methods
Patients and tissues
Samples of thyroid tumors (thyroid hyperplasia, n = 15;
follicular adenoma, n = 9; follicular carcinoma, n = 2;
papillary carcinoma, n = 8; Hashimoto’s disease, n = 3;
Table 1
) were obtained from 46
consecutive unselected patients after surgical resection. Nondegraded
RNA of suitable quality was obtained from 37 of the 46 tissue samples
and used for further analysis. Normal adjacent thyroid tissue was
obtained from 29 of the 37 analyzed cases with thyroid tumors, and
postmortem normal thyroid tissue was obtained from an additional 5
cases (Brain and Tissue Banks for Developmental Disorders, University
of Maryland, Baltimore, MD). Sample aliquots were either immersed in
liquid nitrogen and stored at -70 C or fixed in 10% formalin until
analysis.
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Total RNA was extracted from cell cultures (
3 x
107 cells/group) and excised tissues (after
tissue homogenization) with TRIzol (Life Technologies, Inc., Gaithersburg, MD). RNA derived from JEG-3 choriocarcinoma
cells or rat testis served as a positive control for
PTTG1 expression. Electrophoresed RNA was
transferred to Hybond-N nylon membranes (Amersham International, Little Chalfont, UK). The membrane was
cross-linked, prehybridized (1 h), and hybridized (2 h) at 68 C with
human PTTG cDNA in the presence of 100 µg/ml salmon sperm DNA
(Stratagene, La Jolla, CA). A 900-bp human
PTTG1 cDNA fragment spanning the entire coding
region was labeled with [-32P]deoxy-CTP
using Klenow enzyme (Life Technologies, Inc.). Sodium
iodide symporter (NIS) cDNA was a gift from Nancy Carrasco (Albert
Einstein College of Medicine, New York, NY). Posthybridization washes
were followed by air-drying and autoradiography.
PTTG1 mRNA expression was quantitated using
scanning densitometry, normalized to 18S rRNA expression, and expressed
as the fold increase compared with PTTG1/18S rRNA
ratios in either adjacent normal thyroid tissue (29 of 37) or the mean
PTTG1/18S rRNA ratio measured in 32 normal
thyroid tissues (mean ± 2 SD). The mean ±
SEM PTTG1/18S rRNA ratio in normal
thyroid tissue was 0.56 ± 0.04 arbitrary units.
Western blot analysis
Proteins were prepared from thyroid tissues and cells using RIPA buffer (100 mM NaCl, 0.1% Triton X-100, and 50 mM Tris, pH 8.3) containing a cocktail of enzyme inhibitors (1 mM phenylmethylsulfonylfluoride, 2 µg/ml aprotinin, and 200 µg/ml leupeptin) and denatured (2 min, 100 C) in loading buffer. The protein concentration was determined by the Bradford assay using BSA as a standard. Soluble proteins (50 µg) were separated by electrophoresis in 12% SDS-PAGE gels, transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech), and incubated in 5% nonfat milk in PBS-0.05% Tween solution, followed by incubation with antibodies to PTTG (1:5000) for 24 h at 4 C or with proliferating cytoplasmic nuclear antigen (PCNA) for 2 h at room temperature. After washing in PBS-0.05% Tween (six times, 10 min each time), blots were incubated with appropriate horseradish peroxidase-conjugated anti-IgGs for 1 h at room temperature. After further washes, antigen-antibody complexes were visualized by the ECL chemiluminescence detection system on Hyperfilm ECL (Amersham International).
Immunocytochemistry
Cells cultured on microscope slides in 100-mm plates were fixed in 4% paraformaldehyde, incubated with monoclonal antibody to PCNA (clone PC10, DAKO Corp., Carpinteria, CA), followed by biotinylated secondary antibodies and streptavidin-horseradish peroxidase. Antibody localization was effected using 3,3'-diaminobenzidene tetrachloride. Appropriate positive and negative controls were included.
Animals
Ovariectomized Fischer 344 rats (140–150 g; Harlan Sprague Dawley, Inc., Indianapolis, IN) were housed in a controlled environment (lights on, 0600–1800 h; 22 ± 1 C) with free access to food and water. The use of rats was approved by the institutional animal care and use committee. Subcutaneously implanted osmotic pumps (100 µl; Alzet, Palo Alto, CA), containing 17ß-E2 (1–1000 ng) or 4-hydroxytamoxifen (860 µg) in 90% polyethylene glycol/10% ethanol solution were employed to administer E and/or anti-E (19). Rats were euthanized by CO2 inhalation, thyroid tissues were immersed in liquid N2 and stored at -80 C for RNA and/or protein extraction, and serum was collected for E2 and progesterone assays (20).
Cell culture
Rat FRTL5 cells (American Type Culture Collection, Manassas, VA) were maintained in Coon’s modified F-12 (Life Technologies, Inc.) supplemented with antibiotics, 5% calf serum, and six growth factors as previously described (21). TSH was omitted overnight before TSH induction studies. For primary thyroid cultures, normal thyroid tissue obtained at surgery was minced mechanically and digested for 3 h at 37 C with 10 mg collagenase, 1 mg hyaluronidase, and 1 mg deoxyribonuclease I (Sigma, St. Louis, MO) in 10 ml F-12 medium. Cells were then cultured for 48 h in Coon’s modified medium as described above for 24 h before transient transfection studies (below).
Cell transfection
FRTL-5 cells were transfected with wild-type (Wt) and mutant human PTTG cDNA (13) subcloned in-frame into the mammalian expression vector pCiNeo (Promega Corp., Madison, WI) using Effectene (QIAGEN, Chatsworth, CA), and transfected cells were selected and maintained in medium supplemented with G418 (Geneticin, Life Technologies, Inc.; 1 mg/ml), before screening for mRNA and protein expression. FRTL5 cells transfected with the original pCiNeo vector served as controls, and all experiments were carried out within 20 passages to minimize aging effects in the FRTL5 cells. Primary thyroid cells cultures were transiently transfected using Effectene (QIAGEN, Stanford, Valencia, CA), 48 h after initial culture with Wt-PTTG or vector alone, along with a PSVß-galactosidase expression vector. Measurement of ß-galactosidase expression confirmed equivalent transfection efficiency in vector- and Wt-PTTG transfectants.
Cell proliferation assay
Cell proliferation was measured using CellTiter 96 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide nonradioactive cell proliferation assay according to the manufacturer’s protocol (Promega Corp.). This is a colorimetric assay to determine the number of viable cells based on the cellular conversion of a tetrazolium salt into medium soluble formazan. Absorbance at 490 nm is directly proportional to the number of living cells in culture. Cells (n = 5000) were seeded in 96-well plates (8 wells for each clone in each assay) and incubated at 37 C for 24–72 h. At each time point, dye (20 µl) was added to the wells, and after further incubation at 37 C (4 h), absorbance was recorded at 490 nm using an ELISA reader.
Assays of iodide uptake
125I uptake was measured as previously
described with minor modifications (22). FRTL5 cells
(2 x 105) were seeded into 24-well tissue
culture plates (Costar, Cambridge, MA). Twenty-four hours
after transfection with Wt-PTTG or vector control (primary human
thyroid cultures; n = 3) or 2–3 d after plating (FRTL5 cells),
culture medium was aspirated, and cells were washed with 1 ml HBSS.
Iodide uptake was initiated by adding 500 µl HBSS containing 0.1
µCi carrier-free Na125I and 10 µm/liter
sodium iodide for 60 min at 37 C. The assays were terminated by
aspiration of radioactive medium and washing with 1 ml ice-cold HBSS;
1.0 ml 95% ethanol was added to each well for 1 h and then
transferred to vials before counting in a 
-counter. The DNA content
in each well was measured, and results were expressed as counts per
min/µg DNA. All experiments were performed multiple times (8 wells
for each FRTL5 clone and 3 wells for primary human thyroid cultures) on
2 separate occasions.
PTTG transformation in vitro
For soft agar assay, 60-mm tissue culture plates were coated with 5 ml soft agar (20% 2x F-12, 50% F-12, 10% FBS, and 20% 2.5% agar). Two milliliters of cells suspended in medium were combined with 4 ml agar mixture, and 1.5 ml were added to each plate. Cells were plated (104/dish) and incubated for 14 d, and the number of large colonies (>20 cells) was counted.
Statistical analysis
Results are expressed as the mean ± SEM, and statistical analysis was performed using ANOVA (Bonferroni’s multiple comparison test), taking P < 0.05 as significant.
Results
PTTG overexpression is associated with follicular lesions
Increased PTTG1 mRNA expression was
observed in 10 of 15 cases of thyroid hyperplasia (mean ±
SEM: PTTG1 mRNA,
1.7 ± 0.5-fold increase; P < 0.01, by ANOVA), 7
of 9 follicular adenomas (PTTG1 mRNA,
1.9 ± 0.9-fold increase; P < 0.01), 2 of 2
minimally invasive follicular carcinomas
(PTTG1 mRNA, 2.0 ± 0.4-fold
increase). Modest PTTG increase was noted in 4 of 8 papillary
carcinomas (PTTG1
mRNA, 0.84 ± 0.15-fold increase;
P = NS; Table 1
and Fig. 1, A and B) compared to
mean ± 2 SD
PTTG1 mRNA expression in 32 normal thyroid
tissues. The highest expression (up to 7.2
PTTG1 mRNA fold increase) was observed in
a subset of thyroid hyperplasia, follicular adenomas, and the 2
follicular carcinomas examined (Table 1).
PTTG1 mRNA expression in 3 of 3 cases of
Hashimoto’s disease was similar to that observed in normal thyroid
tissue.
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PTTG contains a proline-rich region containing a PXXP motif near
the C-terminus of the PTTG protein (2). We previously
described the importance of this potential SH3-binding motif in
PTTG-mediated actions and showed that mutation of this proline-rich
(P163A, P170L, P172A, P173L) motif abrogates PTTG-mediated cellular
actions (2). To explore the role of PTTG in thyroid
tumorigenesis, mutant and wild-type PTTG cDNA cloned into a mammalian
expression vector under the control of the cytomegalovirus promoter
were stably transfected into rat FRTL5 cells. Overexpression of
wild-type and mutant PTTG in transfected clones was confirmed by
Northern (Fig. 2A) and Western (Fig. 2B)
blot analyses. Wt-PTTG stable thyroid cell transfectants exhibited
increased bFGF expression compared with vector- or mutant-PTTG
transfectants (Fig. 2C), confirming our previous observations in 3T3
fibroblasts (13). The ability of these cells to undergo
transformation was tested in an anchorage-independent growth assay.
FRTL5 cells overexpressing wild-type PTTG formed numerous large
colonies (44 ± 6 to 72 ± 6 colonies/plate, mean ±
SEM) on soft agar, compared with FRTL5 cells transfected
with vector only (Fig. 2, D and E; 7 ± 1 to 13 ± 1
colonies/plate; P < 0.001). FRTL5 cells expressing
mutant PTTG formed fewer colonies than cells overexpressing wild-type
PTTG and were similar to those observed for cells transfected with
vector alone (ranging from 2 ± 0.3 to 19 ± 1
colonies/plate; P < 0.001). These results demonstrate
in vitro transforming activity of human PTTG and support our
previous observation that a signaling protein(s) containing an SH3
domain(s) mediates PTTG transforming action.
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FRTL5 cells transfected with wild-type PTTG exhibited increased
proliferation rates compared with cells transfected with vector alone
(Fig. 3, A and B; P <
0.001). Cells transfected with PTTG constructs harboring mutations in
the proline-rich region exhibited an intermediate proliferation rate,
which was higher than vector-transfected controls (Fig. 3, A and B;
P < 0.001). In keeping with the enhanced proliferation
of these cells, Western blot demonstrated increased PCNA expression in
wild-type PTTG transfectants (Fig. 3C), and immunohistochemistry
confirmed that some cells exhibited intense PCNA immunostaining (Fig. 3D). We examined Tg levels in the PTTG-transfected cells as a marker of
a differentiated thyrocyte (23). Rat thyrocytes
overexpressing wild-type PTTG expressed lower Tg levels than thyrocytes
transfected with mutant- PTTG or vector alone (Fig. 3E).
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Rat FRTL5 cells overexpressing wild-type
PTTG1 exhibited decreased
125I uptake compared with vector-transfected
controls (Fig. 4A;
P < 0.001). We therefore examined iodide symporter
(NIS) expression in these cells and found that thyrocytes
overexpressing wild-type PTTG1 have
reduced NIS expression compared with vector-transfected controls (Fig. 4B), providing a mechanism for the reduced 125I
uptake. Treatment of parental or vector-transfected FRTL5 cells with
bFGF (5–10 ng/ml) for 48 h led to a 50% decrease in
125I uptake (Fig. 4C; P < 0.001)
similar to PTTG-transfected cells, suggesting that the observed
PTTG-mediated phenotypic alterations are due to secreted bFGF acting in
a paracrine/autocrine manner. Transient overexpression of Wt-PTTG in
normal human thyroid cells in vitro led to a 10% decrease
in 125I uptake (Fig. 4D; P =
0.02), extending and confirming our observations in the stably
transfected FRTL5 cells.
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TSH is an important thyroid growth factor, and the cooperative
regulation of thyrocyte growth by TSH and sex steroids has been
supported by both animal studies and epidemiological analyses
(24, 25). TSH treatment (5–25 µ/ml) of cultured rat
FRTL5 cells or primary human thyroid cells induced pttg
expression in vitro (pttg increase, 5.9- and
2.4-fold, respectively; P = 0.01; Fig. 5, A and B).
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; pttg increase, 5.3-fold).
E-mediated thyroidal pttg induction was abrogated by
coadministration of the anti-E 4-hydroxytamoxifen (860 µg/48 h;
pttg increase, 2.4-fold). Discussion
In a model of thyroid tumorigenesis, a single transformed cell appears to gain a growth advantage due to loss of proliferative regulation. Genomic instability renders the cell prone to large scale chromosomal aberrations, amplifications, deletions, and translocations (1). Allelic deletions are an indication of chromosomal instability, and in several studies of tumor LOH patterns, specific chromosomal regions, such as 3p, 2p, 2q, and 11q, appear more susceptible to allelic loss in thyroid tumors (26, 27, 28, 29, 30, 31). A comprehensive analysis of LOH studies revealed a higher tendency to loose genetic material in follicular neoplasms, particularly follicular carcinomas compared to papillary carcinomas (31), and it was suggested that a fundamental difference may exist for mechanisms controlling chromosomal stability in these two forms of thyroid cancer (31). In this study we demonstrate increased PTTG1 mRNA expression in 10 of 15 thyroid hyperplasias, 7 of 9 follicular adenomas, and 2 of 2 minimally invasive follicular carcinomas, compared with normal thyroid tissue. In contrast, modestly increased PTTG1 mRNA expression was seen in only 4 of 8 papillary carcinomas, and in these tumors, PTTG1 mRNA abundance was lower than in follicular lesions (P = 0.02), although PTTG mRNA expression varied considerably in both follicular and papillary thyroid tumors. As PTTG exhibits cell cycle-dependant expression (32), some of the observed increases in thyroidal tumor PTTG mRNA expression, as with other cell cycle-dependant factors, may in part be secondary to increased mitotic rates, and it is often difficult in a study such as this to discern direct PTTG-mediated effects on the tumor from those secondary to increased proliferation.
As a securin protein, which inhibits sister chromatid separation, PTTG overexpression would inhibit the exit of cells from mitosis, and we have previously demonstrated that transient PTTG overexpression induces both G2/M arrest (32) and apoptosis (33). However, in stable transfectants overexpressing PTTG, as employed in our current studies, we have preselected clones that have survived PTTG-mediated proapoptotic actions and G2/M arrest, presumably due to the acquisition of additional transforming events. These observations indicate that increased PTTG expression in the thyroid tumors is not completely attributable to increased mitotic rates, and that overexpressed or prolonged PTTG expression, by whatever mechanism, during early stage thyroid transformation may lead to dysregulated chromosome separation during mitosis and facilitate the occurrence of LOH events. Overexpressed PTTG may therefore be a key factor in the critical molecular events that determine the development of distinct thyroid tumor phenotypes. Furthermore, the differentially abundant PTTG expression demonstrable in thyroid tumors makes PTTG a potentially important marker of thyroid tumor progression and a target for subcellular thyroid tumor therapies.
Although epidemiological and experimental evidence supports the role of female sex hormones in thyroid neoplasia, it remains unclear whether E and/or androgens are primary tumor promoters. Thyroid cancer is 3 times more common in Caucasian women than in men (1), well differentiated follicular carcinomas occur predominantly in postpubertal and premenopausal women, and ER expression is expressed in about 25% of cases of thyroid carcinoma (24). Here we demonstrate E-mediated induction of thyroidal PTTG expression in vivo. This may be a direct effect or, given that TSH regulates thyroidal PTTG mRNA expression and E administration increases serum TSH (34), may be indirectly mediated via pituitary-secreted TSH. The complex interplay between sex steroids and TSH on thyroid cell growth has been emphasized by animal studies in which both male and female rats were more susceptible to radiation-induced thyroid tumors in the presence of TSH stimulation (25), whereas ovariectomy or castration attenuated thyroid tumor occurrence. These findings suggest that E and a low iodine diet may cooperatively promote pituitary TSH secretion, facilitating TSH-mediated growth and the development of thyroid tumors. PTTG is regulated by E in several tissues, including the thyroid, and also by TSH, indicating the importance of this transforming factor in thyroid tumorigenesis.
125I uptake and Tg are phenotypic markers of a mature thyrocyte (23, 35), and reduced iodide uptake and Tg have been described in thyroid cancers (36, 37) due in part to reduced iodide symporter expression (37). We show here that thyrocytes overexpressing wild-type PTTG1 have reduced NIS expression, reduced 125I uptake, and decreased Tg expression compared with vector-transfected controls, in keeping with a transformed dedifferentiated phenotype, as has been observed after overexpression of other transforming factors in thyroid cells (38, 39). FRTL5 cells overexpressing Wt-PTTG exhibit increased bFGF expression, and bFGF treatment led to reduced 125I uptake, similar to that observed after treatment of FRTL5 cells with other growth factors (40, 41). Thus, PTTG-directed phenotypic alterations in these thyroid cells appear mediated at least in part by secreted bFGF, acting in a paracrine/autocrine manner. bFGF is an important angiogenic factor in many tumor types, including thyroid tumors (42). bFGF stimulates the proliferation of rat FRTL5 thyroid cells (43), which express the high affinity bFGF (Flg) receptor (44), emphasizing bFGF’s role as an important autocrine regulator of thyroid follicular cell growth. Furthermore, TSH induces bFGF expression in cultured thyroid cells in vitro (45) and in experimental models of thyroid hyperplasia, goiter development is associated with concomitant increases in thyroid angiogenesis and follicular bFGF expression (46). We have recently demonstrated PTTG-mediated angiogenesis (47) and previously correlated PTTG expression with vascularity in colorectal tumors (16). Interestingly, a recent report shows E-mediated reduction in NIS expression in rat thyrocytes (48), and here we demonstrate E regulation of thyroid pttg in vivo, and that PTTG overexpression independently reduces NIS expression. Although it is unclear from our studies whether the NIS reduction is a direct effect of E-mediated pttg induction, is a consequence of the pttg-mediated transformed phenotype, or is mediated by secreted bFGF from these transformed cells, E-mediated pttg induction may provide a mechanism for reduced NIS expression in thyroid tumors. These observations integrate the cooperative interactions between hormones, growth factors, and transforming genes, facilitating the thyroid cell transformation process. Given our observed in vitro regulation of thyroidal PTTG mRNA expression by TSH, it would be interesting to examine correlations between PTTG mRNA expression in thyroid tumors and serum TSH levels and/or iodide uptake in these patients.
In conclusion, the transforming factor PTTG is overexpressed in thyroid tumors and is particularly abundant in follicular neoplasms. PTTG overexpression in rat thyrocytes causes cellular transformation and dedifferentiated phenotypic traits. Furthermore, as PTTG is regulated by TSH, E, and bFGF, the sexual discordance observed in thyroid cancer may represent a link between early PTTG-mediated transforming cellular events, such as aneuploidy, and the consequential disruption of growth factor signaling with its attendant phenotypic alterations. We propose that PTTG overexpression in follicular neoplasms leads to increased chromosomal instability, relatively commonly observed in thyroid follicular lesions, but infrequent in papillary thyroid cancers. Downstream targets of PTTG therefore appear implicated in the divergence of follicular vs. papillary thyroid cancer phenotypes.
Acknowledgments
We thank Shlomo Melmed for his considerable contribution and critical reading of the manuscript, and Nancy Carrasco for the gift of rat NIS cDNA.
Footnotes
This work was supported by grants from Thyroid Research Advisory Council (TRAC) (to A.P.H.) and the Doris Factor Molecular Endocrinology Laboratory.
Abbreviations: bFGF, basic fibroblast growth factor; NIA, sodium iodide symporter; PCNA, proliferating cytoplasmic nuclear antigen; PTTG, pituitary tumor transforming gene; Wt, wild type.
Received February 1, 2001.
Accepted June 6, 2001.
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) and 48 h (
) are shown for
individual clones (control, n = 2; Wt-PTTG, n = 3; mutant
PTTG, n = 3) in multiplicate (n = 8) as the mean ±
SEM and compared by ANOVA. Western blot (C) and
immunocytochemical analysis (D) demonstrate increased PCNA and
decreased Tg (E) expression in Wt-PTTG-transfected FRTL5 cells compared
with vector- and/or mutant-PTTG-transfected controls.
