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Endocrinology, doi:10.1210/en.2006-0544
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Endocrinology Vol. 147, No. 10 4781-4791
Copyright © 2006 by The Endocrine Society

Pituitary Tumor Transforming Gene Overexpression Facilitates Pituitary Tumor Development

Ines Donangelo, Shiri Gutman, Eva Horvath, Kalman Kovacs, Kolja Wawrowsky, Michael Mount and Shlomo Melmed

Department of Medicine (I.D., S.G., K.W., M.M., S.M.), Cedars-Sinai Research Institute, David Geffen School of Medicine at University of California Los Angeles, Los Angeles, California 90048; and St. Michael’s Hospital (E.H., K.K.), Toronto, Ontario, Canada M5B 1W8

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: melmeds{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Intrinsic and extrinsic stimuli result in profound pituitary growth changes ranging from hypoplasia to hyperplasia. Pituitary tumor transforming gene (PTTG) abundance correlates with pituitary trophic status. Mice with Pttg inactivation exhibit pituitary hypoplasia, whereas targeted pituitary PTTG overexpression driven by {alpha}-subunit glycoprotein ({alpha}GSU) promoter results in focal pituitary hyperplasia. To test the impact of pituitary hyperplasia on tumor development, we crossbred {alpha}GSU.PTTG with Rb+/– mice, which develop pituitary tumors with high penetrance. Pituitary glands of resulting bitransgenic {alpha}GSU.PTTGxRb+/– mice were compared with monotransgenic {alpha}GSU.PTTG, Rb+/–, and wild-type mice. Confocal microscopy showed that PTTG-overexpressing cells have enlarged nuclei and marked redistribution of chromatin, and electron microscopy of {alpha}GSU.PTTG pituitaries showed enlarged gonadotrophs with prominent Golgi complexes and numerous secretory granules. These morphological findings were even more remarkable in {alpha}GSU.PTTGxRb+/– pituitaries. Mice from all four genotypes were sequentially imaged by magnetic resonance imaging to evaluate pituitary volume, and glands from {alpha}GSU.PTTGxRb+/– mice were the largest as early as 2 months of age (P = 0.0003). Cumulative incidence of pituitary tumors visualized by magnetic resonance imaging did not differ between Rb+/– and {alpha}GSU.PTTGxRb+/– mice. However, anterior lobe tumors determined after necropsy were 3.5 times more frequent in {alpha}GSU.PTTGxRb+/– than in Rb+/– mice (P = 0.0036), whereas the frequency of intermediate lobe tumors was similar. In summary, {alpha}GSU.PTTGxRb+/– pituitary glands exhibit enhanced cellular activity, increased volume, and higher prevalence of anterior pituitary tumors, indicating that changes in pituitary PTTG content directly relate to both pituitary trophic status and tumorigenic potential.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
DESPITE ADVANCES IN understanding the pathogenesis of pituitary tumors, the primary etiology of these common adenomas remains enigmatic. Pituitary tumor formation and progression occurs in a relatively diverse background of chromosomal instability, epigenetic changes, and cell mutations (1, 2, 3). Compared with the normal pituitary gland, a wide range of gene expression profiles is observed between the different pituitary adenomas subtypes (4, 5). With the exception of the Gs{alpha} mutation found in approximately one third of GH-secreting adenomas (6, 7, 8, 9), there are few clearly defined proximal pathways for human pituitary tumorigenesis.

Extrinsic and intrinsic signals subserve remarkable pituitary gland plasticity, with changes in pituitary size ranging from hypoplasia to hyperplasia (10). In animal models, pituitary trophic status correlates with the likelihood of tumor development because hyperplastic pituitary glands frequently develop adenomas, suggesting that sustained long-term pituitary hyperplasia ultimately results in tumor formation (11, 12). At the opposite end of the spectrum, hypoplasia likely protects the pituitary from tumor development. In mouse models with combined hypoplastic and tumorigenic genetic abnormalities, tumor incidence is lower than in mice harboring the tumorigenic genetic change alone. This pattern is observed in both Pttg–/–Rb+/– mice (inactivation of pttg leads to pituitary hypoplasia, and Rb disruption causes pituitary tumor) and in double-mutant p18Ink4c–/–cdk4–/– mice (p18 inactivation causes pituitary hyperplasia that progresses to tumor, and loss of cdk4 results in pituitary hypoplasia) (13, 14). Understanding mechanisms that regulate pituitary plasticity therefore provides insights for disrupting development and progression of pituitary tumors.

Pituitary tumor transforming gene (PTTG) was isolated from a pituitary tumor cell line, and its overexpression results in cellular transformation in vitro and tumor formation in nude mice (15). PTTG was identified as the index mammalian securin, regulating sister chromatid separation during mitosis (16), and excessive or suppressed PTTG levels result in aneuploidy (17, 18). PTTG abundance correlates with pituitary gland plasticity. Transgenic mouse models of both PTTG overexpression and inactivation support a causal role for changes in PTTG levels on development of pituitary hyperplasia and hypoplasia and on conferring tumor growth advantage or tumor growth protection, respectively (Table 1Go). Mice with transgenic expression of human PTTG1 driven by the {alpha}-subunit glycoprotein ({alpha}GSU) promoter express PTTG in LH-, FSH-, TSH-, and GH-secreting cells (19). {alpha}GSU.PTTG mice develop histopathological pituitary findings ranging from frequent hyperplasia to occasional microadenomas. Increased serum LH, testosterone, GH, and IGF-I levels result in marked seminal vesicle and prostate enlargement in male {alpha}GSU.PTTG mice. In contrast, Pttg inactivation results in opposite trophic effects, i.e. pituitary, pancreatic ß-cell, splenic, and testicular hypoplasia (18).


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  		TABLE 1. Pituitary phenotype in murine models of PTTG overexpression ({alpha}GSU.PTTG) or inactivation (pttg–/–)

 
To further understand the role of PTTG on pituitary tumor development, {alpha}GSU.PTTG mice were crossbred with Rb+/– mice, a well-established mouse model for pituitary tumors (20). The pituitary glands of the resulting bitransgenic {alpha}GSU.PTTGxRb+/– mice exhibited marked hyperplasia and higher prevalence of anterior lobe pituitary tumors, compared with singly transgenic {alpha}GSU.PTTG or Rb+/– mice. These findings indicate that changes in PTTG abundance correlate with pituitary tumorigenic potential.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
This study was approved by the Institutional Animal Care and Use Committee and was performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Mice of a B6C3 genetic background harboring the P{alpha}GSU-PTTG1-IRES-eGFP ({alpha}GSU.PTTG) transgene were previously described (19). Rb+/– mice on a 129/Sv genetic background were purchased from The Jackson Laboratory (Bar Harbor, ME) and backcrossed for four generations to a C57BL/6 parental genotype. {alpha}GSU.PTTG mice were crossbred with Rb+/– mice for two generations, resulting in offspring with the following genotypes: wild type (WT), {alpha}GSU.PTTG, Rb+/–, and bitransgenic {alpha}GSU.PTTGxRb+/–. Animals were genotyped by PCR for the presence of enhanced green fluorescent protein (eGFP) (19) and Rb mutation (20) as previously described. Phenotype analysis was performed by comparing litter mates with different genotypes.

Bitransgenic {alpha}GSU.PTTGxRb+/– mice and age- and sex- matched controls ({alpha}GSU.PTTG, Rb+/–, and WT) were euthanized using CO2 chambers. Blood was withdrawn directly from the heart, pituitary glands were collected and weighed, and other organs were carefully inspected and collected for analysis if abnormal.

Morphological methods
For light microscopy, the pituitaries were fixed in 10% buffered formalin, paraffin embedded, sectioned 4 microns thick, and stained with hematoxylin and eosin. Sections were also analyzed by double-label immunohistochemistry as previously described (19), using the antibodies: rabbit antihuman ACTH (1:200), rabbit antirat GH (1:800), rabbit antirat TSHß (1:200), guinea pig antirat prolactin (PRL) (1:200), guinea pig antirat LHß (1:200), and guinea pig antirat {alpha}-subunit (1:200) (National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD). Secondary antibodies were Alexa Fluor 488 antiguinea pig and Alexa Fluor 568 antirabbit (Molecular Probes, Carlsbad, CA). Slides were counterstained with 4,6-diamidino-2-phenylindole to visualize nuclei.

PCNA nuclear staining was performed according to instructions provided by the kit (Zymed Laboratories, Inc., San Francisco, CA), and index of stained nuclei was determined by a rater (S.G.) blinded for genotype with Image J cell counter (21).

For electron microscopy, small pituitary pieces were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, postfixed in 1% osmium tetroxide, processed through graded series of ethanol, then propylene oxide, and embedded in Araldite. Semithin sections stained with toluidine blue, and ultrathin sections with uranyl acetate and lead citrate were studied with a Philips 410-LS electron microscope (Phillips Electronic Instruments, Rahway, NJ).

Confocal microscope images were obtained using a TCS-SP confocal scanner (Leica Microsystems, Mannheim, Germany). Before imaging, pituitary samples were fixed in 10% buffered formalin for 2 h and then incubated in 2 µM TO-PRO-3 (Molecular Probes) and 50 µM phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) (Sigma-Aldrich, St. Louis, MO) overnight for nucleic acid and actin filament staining, respectively. A Plan Apo 63 x 1.2 numerical aperture lens was used, and the dimensions of the confocal stacks were 150 x 150 µm and 10–15 µm deep. To excite the fluorescent dyes, three different lasers were used in sequential acquisition mode: 488-nm line of argon laser for eGFP, 568-nm line of argon-krypton laser for phalloidin-TRITC, and 633-nm line of helium-neon laser for TO-PRO-3. Cells expressing {alpha}GSU.PTTG were promptly identified due to presence of eGFP signal. Images from different fields of the pituitary were obtained, and analysis of nuclear morphology was performed without knowledge of eGFP status, i.e. the green layer of the image was hidden using Adobe Photoshop 5.5 (Adobe Systems, San Jose, CA) software. Nuclei were considered altered if they were clearly different from the average WT pituitary cell (Fig. 1aGo), i.e. if the nucleus was markedly enlarged (Fig. 1bGo) or showed diffuse or peripheral accumulation of nucleic acid (Fig. 1Go, b and c, respectively) as opposed to the presence of dense and mainly central heterochromatin foci in WT nuclei (Fig. 1aGo). Nuclei that could not be undoubtedly classified as normal or altered were excluded from evaluation, and this was the case in 14% (35 of 248) of WT, 17% (54 of 314) of {alpha}GSU.PTTG, 24% (69 of 291) of Rb+/–, and 24% (61 of 256) of {alpha}GSU.PTTGxRb+/– analyzed cells. After classification of nuclear morphology, eGFP status was revealed, and the nuclear morphology profile from cells overexpressing PTTG (eGFP positive) or not (eGFP negative) was contrasted.


Figure 1
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  		FIG. 1. Confocal microscopy images of pituitary nuclei used as reference for classification into normal or altered morphology. a, Normal-sized nucleus with habitual chromatin distribution, i.e. with scattered foci of heterochromatin. b, Unusually enlarged nucleus with very few heterochromatin foci located close to the nuclear membrane. c, Nucleus with altered distribution of chromatin concentrated in the nuclear periphery, forming a ring-shaped pattern. Nucleic acid is stained with TO-PRO-3 (blue), and actin filaments were stained with phalloidin-TRITC (red) to help define cell boundaries.

 
Magnetic resonance imaging (MRI)
Pituitary glands were imaged in vivo with MRI, as previously described (19), in eight WT, 10 {alpha}GSU.PTTG, nine Rb+/–, and 10 bitransgenic {alpha}GSU.PTTGxRb+/– mice with similar proportion of males and females, starting at 2 months of age, and then every 2 months until 10 months. Briefly, mice were anesthetized with ip injections of ketamine (70 µg/g) and medetomidine (1 µg/g) and imaged using a small solenoidal receiver coil. Atipamezole (1 µg/g ip) was used to reverse the effect of medetomidine. The pituitary area from each sagittal plane was determined by manually drawing a line along the edge of the gland in a magnified image. Two independent raters (S.G. and M.M.) blinded for mice genotype performed this measurement. Pituitary volume was determined by adding the number of pixels within the pituitary gland of all the sagittal planes.

Statistical analysis
Statistical calculations and significance tests were performed using the statistical software package SAS version 9.1 (SAS Institute, Cary, NC). Reproducibility of pituitary volume measurement by MRI was determined as follows: in nine mice, the pituitary was imaged three consecutive times, repositioning the animals between each imaging. The mean absolute percent difference in pituitary volume between three different images was 5.47, ranging from 0.5–13.7%. Changes of pituitary volume across time were assessed by repeated measures ANOVA (RMANOVA) with time (age) as the within-subjects factor (at four levels: 2, 4, 6, and 8 months) and genotype as the between-subjects factor (at four levels: WT, Rb+/–, {alpha}GSU.PTTG, and {alpha}GSU.PTTGxRb+/–). Because volumes should be related across time, an autoregressive covariance structure was assumed for the RMANOVA. Genotype differences in mean volume at a specific time were assessed by one-way ANOVA; post hoc comparisons were performed by Dunnett’s t test with WT as the control group. Frequency of intermediate lobe and anterior lobe pituitary tumors in Rb+/– and {alpha}GSU.PTTGxRb+/– mice was compared by Fisher’s exact test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Electron microscopy: PTTG overexpression results in prominent Golgi activity and increase number of secretory granules
Ten pituitaries derived from all the genotypes were analyzed by electron microscopy (3- to 5-month-old males and females). Ultrastructure of cell types in pituitaries derived from {alpha}GSU.PTTG mice was normal, with the exception of gonadotrophs, as shown in Fig. 2BGo. These cells were slightly to moderately enlarged and appeared more numerous when compared with WT specimens (Fig. 2AGo). Gonadotrophs exhibited unusually abundant secretory granules and prominent, very active Golgi complexes (Fig. 2BGo). Occasional cells not typical of pituitary cell types were encountered; these were large, lucent, and exhibited secretory granules of variable sizes and prominent Golgi complexes, suggesting altered or differentiating gonadotrophs, as shown in Fig. 2CGo (arrowhead).


Figure 2
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  		FIG. 2. Electron microscopy images of mice pituitary glands. A, WT specimen depicting a normal gonadotroph cell including populations of small and large secretory granules. Gonadotrophs are ovoid cells that exhibit well-developed RER, ring-shaped Golgi complex, and two different sizes of secretory granules. Part of a PRL cell is shown as well (bottom). Magnification, x13,200. B, Monotransgenic {alpha}GSU.PTTG pituitary specimen showing portions of three hyperplastic gonadotrophs. The secretory granules are more numerous than usual; the RER is well developed, but not dilated. The other cell type shown is a lactotroph (right). Magnification, x13,200. C, Bitransgenic {alpha}GSU.PTTGxRb+/– specimen with marked gonadotroph hyperplasia; both small and large secretory granules are abundant, the Golgi complex is prominent, and the RER shows normal morphology. Top, Part of an unidentified cell with lucent cytoplasm and minute, sparse, peripherally disposed secretory granules is depicted (arrowhead). Magnification, x9000. D, {alpha}GSU.PTTGxRb+/– specimen with aggregates of glycogen ß particles (arrow) not known to normally occur are evident within the cytoplasm of a hyperplastic gonadotroph. Magnification, x21,300. G, Golgi complex.

 
Electron microscopy of pretumorous pituitary glands derived from Rb+/– mice did not show obvious abnormalities. A pituitary tumor sample derived from an 11-month-old Rb+/– female showed a cell population with low-grade functional differentiation, with numerous small vacuolated rough endoplasmic reticulum (RER) profiles, as well as a well-developed Golgi complex containing occasional immature granules. Light microscopy and immunostaining for pituitary hormones of this tumor sample identified it as an ACTH-positive intermediate lobe tumor (data not shown).

Pituitary glands derived from {alpha}GSU.PTTGxRb+/– mice exhibited heterogeneous cell morphology, ranging from areas with large, lucent cells with minute secretory granules (presumably altered or differentiating gonadotrophs) similar to those observed in {alpha}GSU.PTTG pituitaries (Fig. 2CGo, arrowhead), to massive gonadotroph hyperplasia. The ultrastructural appearance of hyperplastic gonadotroph cells shows several small (150–350 nm) and many larger (over 350 nm) secretory granules, but no proliferation or dilatation of RER (Fig. 2CGo), the salient feature of so-called castration cells. Notably, glycogen ß-particles were present in the cytoplasm of several gonadotrophs (Fig. 2DGo). In addition, small areas of hyperplastic thyrotroph cells were also noted. These cells are polyhedral or elongated and exhibit well-developed and dilated RER, but without the so-called thyroidectomy changes. Two distinct morphological tumor types were observed in a pituitary derived from a 5-month-old {alpha}GSU.PTTGxRb+/– male; one tumor subtype was probably of gonadotroph origin (Fig. 3AGo), whereas the second tumor was likely derived from a thyrotroph cell (Fig. 3BGo). Pituitary tumors derived from two {alpha}GSU.PTTGxRb+/– females (12 and 13 months old) were also analyzed. Frank neoplasia likely of gonadotroph origin and areas of gonadotroph and thyrotroph hyperplasia similar to those found in younger {alpha}GSU.PTTGxRb+/– mice were observed.


Figure 3
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  		FIG. 3. Ultrastructural appearance of a bitransgenic {alpha}GSU.PTTGxRb+/– pituitary specimen with two distinct neoplasms. A, one presumably of gonadotroph derivation; features of uniform ovoid nuclei, lucent cytoplasm, paucity of organelles and sparse, minute, peripheral secretory granules resemble those of the unidentified cell type (see Fig. 2CGo). Magnification, x4800. B, Second tumor, likely of thyrotroph origin, features large irregular cells with markedly irregular nuclei. The sizable, low-density cytoplasm is loosely filled with vesicular RER, and small secretory granules are noted only within the Golgi region (arrows). Magnification, x4800.

 
Confocal microscopy: PTTG overexpression results in chromatin reorganization
Pituitaries from 2-month-old WT, Rb+/–, monotransgenic {alpha}GSU.PTTG, and bitransgenic {alpha}GSU.PTTGxRb+/– females from the same litter were imaged with confocal microscopy. A representative confocal image from {alpha}GSU.PTTG pituitary can be appreciated in Fig. 4Go, where distinct nuclear morphology from cells overexpressing PTTG transgene (eGFP signal) can be promptly contrasted with the normal (eGFP negative) nuclear morphology. Pituitary cells were individually analyzed by studying their whole extent through the several confocal layers. Figure 5Go shows representative samples of pituitary cell nuclei observed in the four genotypes, and nuclear morphology analysis is summarized in Table 2Go. In WT pituitary, 9% (19 of 213) of nuclei were classified as altered. In Rb+/– pituitary, the frequency of altered nuclei increased to 28% (62 of 222) mainly due to the presence of nuclei with diffuse chromatin distribution (poorly defined heterochromatin) (Fig. 5Go, j–n) because macronuclei were seldom observed in this genotype. The cell population in both monotransgenic {alpha}GSU.PTTG and bitransgenic {alpha}GSU.PTTGxRb+/– pituitaries was divided into eGFP-negative (normal Pttg expression) and eGFP-positive (cells expressing PTTG transgene). Within the eGFP-negative cells, the frequency of altered nuclei was 23% (10 of 43) and 14% (13 of 93) in {alpha}GSU.PTTG and {alpha}GSU.PTTGxRb+/–, respectively. On the other hand, nuclei in pituitary cells overexpressing PTTG (eGFP positive) were generally altered. Eighty-eight percent (192 of 217) of eGFP-positive cells in monotransgenic {alpha}GSU.PTTG (Fig. 5Go, f–i) and 95% (97 of 102) of eGFP-positive cells in bitransgenic {alpha}GSU. PTTGxRb+/– pituitaries (Fig. 5Go, o–r) exhibited altered nuclei. In addition to large nuclei and redistribution of chromatin, a fraction (~15%) of altered nuclei in bitransgenic {alpha}GSU.PTTGxRb+/– pituitary glands also exhibited large areas within the nuclei that did not stain with TO-PRO-3 (Fig. 5Go, o and p). These large nuclear vacuoles were also noted less frequently in cells from {alpha}GSU.PTTG pituitary.


Figure 4
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  		FIG. 4. Overview of pituitary cells expressing {alpha}GSU-PTTG1-IRES-eGFP transgene. a and b, Duplicates of the same image, where a is the untouched image, and in b, the green layer (eGFP) has been hidden for better visualization of nuclear morphology. Contrast between eGFP positive (overexpressing PTTG) and eGFP negative (normal PTTG content) can be appreciated, notably presence of macronuclei and reorganization of chromatin.

 

Figure 5
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  		FIG. 5. Confocal microscopy images of cells from WT, monotransgenic {alpha}GSU.PTTG, Rb+/–, and bitransgenic {alpha}GSU.PTTGxRb+/– pituitaries. Representative samples of WT (a–e) pituitary cells with oval-shaped nuclei and readily identified foci of condensed chromatin. The transgene expressing PTTG is tagged with eGFP; therefore, in {alpha}GSU.PTTG and {alpha}GSU.PTTGxRb+/– pituitaries, cells overexpressing PTTG were identified by the presence of eGFP (f–i and o–r, top images). Upon removal of green layer (f–i and o–r, bottom images), altered nuclei were visible: macronuclei (f and o–r, bottom), redistribution of chromatin, i.e. loss of central foci of heterochromatin (f–i and o–r, bottom) and accumulation of chromatin adjacent to the nuclear membrane (g–i, bottom). In a fraction of Rb+/– pituitary cells, heterochromatin foci were not identified (j–n), and this chromatin distribution pattern was considered altered. Nucleic acid is stained with TO-PRO-3 (blue) and actin filament with phalloidin-TRITC (red).

 

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  		TABLE 2. Nuclear morphology according to genotype and eGFP status

 
{alpha}GSU.PTTGxRb+/– mice exhibit marked pituitary enlargement
Pituitary volume was assessed by MRI in bitransgenic {alpha}GSU.PTTGxRb+/– mice and compared with WT, monotransgenic {alpha}GSU.PTTG, and Rb+/– mice starting at 2 months of age and then every 2 months for 10 months. Volume assessment by this method allowed comparison of pituitary enlargement caused by hyperplasia among the different genotypes and distinguished pituitary growth caused by hyperplasia vs. adenoma. To assess information on pretumorous pituitary growth, mice exhibiting tumors visualized by MRI were excluded. The RMANOVA pituitary volume model was highly significant (P < 0.0001), and mean pituitary volumes (averaging across time) differed by genotype (P = 0.016). As shown in Fig. 6Go, at 2 months of age, bitransgenic {alpha}GSU.PTTGxRb+/– mice had increased pituitary volumes compared with WT pituitary volumes (234 ± 39 vs. 179 ± 20 pixels, P = 0.0003; ANOVA). Bitransgenic {alpha}GSU.PTTGxRb+/– mice also exhibited the largest pituitary volumes at 4, 6, and 8 months; however, the difference from other genotypes was not significant at these times. The pattern of pituitary growth over time was similar between sexes (data not shown).


Figure 6
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  		FIG. 6. Bitransgenic {alpha}GSU.PTTGxRb+/– mice exhibit significantly larger pituitaries across genotypes (P = 0.016). Shown are average pituitary gland volumes as assessed by MRI in WT, {alpha}GSU.PTTG, Rb+/–, and {alpha}GSU.PTTGxRb+/– mice, at ages 2, 4, 6, and 8 months. Two pituitaries from the {alpha}GSU.PTTGxRb+/– group (at 6 and 8 months) and 3 pituitaries from the Rb+/– group (1 at 4 months and 2 at 8 months) were excluded from the cohort for volume determination because they exhibited tumors. Over the study, one {alpha}GSU.PTTG, one Rb+/–, and 3 {alpha}GSU.PTTGxRb+/– mice died due to anesthesia-related complications. Hence, the number of pituitaries analyzed at 2, 4, 6, and 8 months was 10, 10, 9, and 9 in {alpha}GSU.PTTG; 9, 7, 7, and 5 in Rb+/–; 10, 10, 8, and 5 in {alpha}GSU.PTTGxRb+/–, respectively; and 8 for all ages in WT mice. Mean ± SE of the pituitary volume in pixels at 2, 4, 6, and 8 months was 179 ± 7, 200 ± 8, 199 ± 10, and 218 ± 9 in WT; 203 ± 9, 219 ± 8, 214 ± 15, and 237 ± 11 in {alpha}GSU.PTTG; 199 ± 7, 195 ± 9, 210 ± 12, and 221 ± 21 in Rb+/–; and 234 ± 12, 240 ± 17, 225 ± 11, and 241 ± 13 in {alpha}GSU. PTTGxRb+/– mice, respectively.

 
Pituitary tumor development assessed by MRI
Distinct pituitary tumors were detected by MRI in Rb+/– and bitransgenic {alpha}GSU.PTTGxRb+/– mice but not in WT or monotransgenic {alpha}GSU.PTTG mice. Pituitary tumors were identified between 1 and 4 months before animals became visibly ill, allowing follow-up of tumor growth, as depicted in Fig. 7Go. Cumulative pituitary tumor incidence until age 10 months as assessed by MRI was not different between Rb+/– and {alpha}GSU.PTTGxRb+/– mice (Fig. 8AGo); however, qualitative differences were noted when comparing tumors from both genotypes. Two mice died before 10 months due to tumor mass, and they were maintained in the cumulative pituitary tumor incidence analysis because their tumors had been detected by MRI.


Figure 7
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  		FIG. 7. Pituitary tumor onset and progression assessed by MRI. Images consist of MIP of reformatted data of pituitary gland of the same bitransgenic {alpha}GSU.PTTGxRb+/– mouse at 4, 6, 8, and 10 months of age. Star (hyperintense signal), Normal pituitary. Arrow (hypointense signal), pituitary tumor.

 

Figure 8
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  		FIG. 8. Bitransgenic {alpha}GSU.PTTGxRb+/– mice exhibit higher prevalence of anterior lobe and similar prevalence of intermediate lobe pituitary tumors when compared with Rb+/– mice. A, Similar cumulative incidence of pituitary tumors in Rb+/– and {alpha}GSU. PTTGxRb+/– mice determined in vivo by MRI. n, Total number of animals that had pituitary gland imaged until 10 months of age or that had visible tumor in MRI. B, Pathological analysis of pituitary tumors reveals that frequency of tumors arising from anterior lobe is higher in {alpha}GSU.PTTGxRb+/– (white bars) than in Rb+/– (black bars) pituitary tumors (**, P = 0.0036), but frequency of tumors arising from the intermediate lobe is similar. n, Total number of pituitary tumors analyzed.

 
Pituitary tumor pathology
Histopathological analyses of the first pituitary tumors derived from 20 bitransgenic {alpha}GSU.PTTGxRb+/– and 20 Rb+/– mice were compared. The average age of pituitary tumor harvest was similar for both genotypes [10.7 (from 6–14) months in {alpha}GSU.PTTGxRb+/– mice and 10.9 (from 5–15) months in Rb+/– mice], and weight was also comparable (mean ± SEM, 61 ± 13 mg in {alpha}GSU.PTTGxRb+/– and 57 ± 17 mg in Rb+/–). Pituitary tumors originating from the anterior lobe were distinguished from those originating from intermediate lobe by light microscopy and immunohistochemistry for pituitary hormones. Intermediate lobe tumors immunostain for ACTH only, whereas anterior lobe tumors immunostain for one or more of the anterior lobe hormones. Immunostaining profile of the pituitary tumors is listed in Table 3Go. Of note, in both genotypes, a subset of pituitary tumors comprised two distinct neoplasms, i.e. both anterior and intermediate lobe tumors coexisting in the same sample. In almost all samples, only one anterior lobe and/or one intermediate lobe tumor were observed. As indicated in Table 3Go, the only exception was sample 2, derived from a Rb+/– mouse that exhibited two distinct small anterior lobe tumors. Eighty percent (16 of 20) of the pituitary tumors derived from Rb+/– mice originated from the intermediate lobe only, 10% (two of 20) from the anterior lobe only, and in two cases (10%), the pituitary exhibited tumors arising in both intermediate and anterior lobes. Thirty percent (six of 20) of pituitary tumors in {alpha}GSU.PTTGxRb+/– mice were of intermediate lobe origin only, 25% (five of 20) arose exclusively in the anterior lobe, and 45% (nine of 20) of pituitary tumors from bitransgenic genotype mice had adenomatous foci in both intermediate and anterior lobes. As depicted in Fig. 8BGo, the frequency of tumors arising from the anterior lobe was over 3.5-fold higher in bitransgenic {alpha}GSU.PTTGxRb+/– compared with Rb+/– pituitary tumors [70% (14 of 20) vs. 20% (four of 20), respectively; P = 0.0036]. The frequency of intermediate lobe tumors was similar in {alpha}GSU.PTTGxRb+/– and Rb+/– samples [75% (15 of 20) vs. 90% (18 of 20), respectively; P = 0.41].


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  		TABLE 3. Rb+/– and {alpha}GSU.PTTGxRb+/– pituitary tumor hormone immunostaining profile

 
Tumors were derived from females in 12 of the 20 {alpha}GSU.PTTGxRb+/– and eight of the 20 Rb+/– pituitary tumor samples. No sex difference in age or tumor weight was noted between genotypes. Bitransgenic {alpha}GSU.PTTGxRb+/– females exhibited slightly higher prevalence of anterior lobe tumors (10 of 12 tumors) compared with males with the same genotype (four of eight pituitary tumors). No sex difference in the prevalence of anterior lobe tumors was noted in Rb+/– mice (one of eight in females and three of 12 in males) or in the prevalence of intermediate lobe pituitary tumors in both genotypes.

To assess whether anterior lobe pituitary tumors from {alpha}GSU.PTTGxRb+/– and Rb+/– mice proliferate at different rates, we compared their PCNA staining index. The percentage of PCNA positive cells was similar in anterior lobe tumors from both genotypes (69 ± 17% vs. 73 ± 24% in {alpha}GSU.PTTGxRb+/– and Rb+/–, respectively) (Fig. 9Go).


Figure 9
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  		FIG. 9. Anterior lobe pituitary tumors from {alpha}GSU.PTTGxRb+/– and Rb+/– mice have similar proliferation rate. The percentage of PCNA stained cells is similar in {alpha}GSU.PTTGxRb+/– and Rb+/– anterior lobe pituitary tumors. n, Total number of pituitary tumors. AL, Anterior lobe.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Pituitary trophic status may directly correlate with the likelihood of tumor development. It is, however, difficult to test this relationship in serial human specimens. Pituitary tumors are essentially benign and occasionally are incidentally found during autopsies or imaging examinations. The prevalence of pituitary adenomas in autopsy studies ranges from 3–27% (22, 23, 24, 25, 26). Clinically active pituitary tumors are notably less common, with a reported prevalence of <1% (27). In a large survey including 506 patients harboring pituitary incidentalomas, the most frequent diagnosis was nonfunctioning pituitary adenoma (28). Pituitary hyperplasia or hypoplasia are not commonly reported in autopsy studies, likely due to diagnostic difficulties. Hyperplasia is defined as an abnormal increase in cell number, and may have a diffuse or nodular pattern. Morphology and anatomical distribution differs between hyperplasias of individual pituitary cell types (29). Pituitary hypoplasia, i.e. a small gland with decreased cell number, is infrequently encountered and usually results from genetic abnormalities linked to pituitary hormone deficiency.

Common progression of pituitary hyperplasia to neoplasia is not supported by studies of human tumor specimens. Physiological pituitary hyperplasia (pregnancy, lactation), pituitary enlargement due to estrogen administration, or untreated primary hypothyroidism are infrequently associated with adenoma development (29, 30, 31, 32). Although true GH- secreting adenoma development in patients with ectopic GHRH syndrome has been reported (33, 34), patients harboring GHRH-producing tumors usually develop acromegaly due to somatotroph hyperplasia (34, 35). Surgically removed pituitary adenomas are often surrounded by nonhyperplastic pituitary tissue and are generally monoclonal (36, 37). However, animal models clearly show that sustained long-term pituitary hyperplasia ultimately results in tumor progression (11, 12). Whether or not sustained (several years) pituitary hyperplasia in humans results in a similarly higher incidence of neoplasia development is presently unknown. Cellular changes preceding, or not related to, gross morphological abnormalities may have a permissive effect on pituitary adenoma development.

Because PTTG abundance correlates with pituitary gland plasticity, regulation of this gene may subserve a mechanism for affecting tumor formation. Pttg knockout results in pituitary hypoplasia (18, 19), and Pttg inactivation in Rb+/– mice results in relative protection from tumor development (13). Although Rb+/– mice have a cumulative pituitary tumor incidence of 86% by 13 months, only 30% of bitransgenic Pttg–/–Rb+/– mice develop pituitary tumors by the same age (P < 0.01) (13). Targeted PTTG overexpression in mice driven by the {alpha}GSU promoter results in focal pituitary hyperplasia with hormone hypersecretion (19). In this study, we extend these insights, showing that combined Rb+/– and targeted pituitary PTTG overexpression enhances pituitary hyperplasia and tumor prevalence. Anterior lobe pituitary tumors arose relatively infrequently in Rb+/– mice, representing only 20% of tumors. Other studies on Rb+/– mice reported a prevalence of anterior lobe tumors ranging from 7–65%, and this variability is attributed to different genetic backgrounds (38, 39, 40). Bitransgenic {alpha}GSU.PTTGxRb+/– mice have enlarged pituitary glands and 3.5-fold increase in the frequency of tumors originating from {alpha}-subunit-expressing cells. A proposed depiction of effects resulting from in vivo changes in pituitary PTTG content is shown in Fig. 10Go.


Figure 10
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  		FIG. 10. Pituitary PTTG content correlates with gland plasticity and with tumor formation potential. On the left side of the inverted triangle are listed mouse models with descending pituitary PTTG content, with or without the combination with tumorigenic Rb+/–. The horizontal bars composing the inverted triangle represent the observed effects of the different genotypes on pituitary trophic status, which correlates with pituitary tumorigenic potential (arrow). [Modified from Ref. 55 .]

 
PTTG overexpression permits a proliferative advantage, and this effect is enhanced by the combination with heterozygous Rb inactivation. By MRI analysis, we show that {alpha}GSU.PTTGxRb+/– mice exhibit significantly larger nontumorous pituitary glands compared with control litter mates. This enlargement was remarkable at 2 months of age, becoming less pronounced in the older age groups. When pituitary glands developed tumors, they were excluded from the cohort for volume determination, and this likely explains the attenuation of volume difference between {alpha}GSU.PTTGxRb+/– pituitaries and the other genotypes in the older age groups. Bitransgenic {alpha}GSU.PTTGxRb+/– mice with larger pituitary glands at age 2 months were more likely to develop tumors. Pituitary volume (mean ± SEM) was 293 ± 24 in animals that developed pituitary tumors earlier than 8 months vs. 255 ± 18 pixels in those that developed tumors after 8 months (P = not significant). Pituitary enlargement in monotransgenic {alpha}GSU.PTTG and bitransgenic {alpha}GSU.PTTGxRb+/– mice is likely accounted for by hyperplasia of the anterior lobe, specifically of {alpha}GSU-positive cells overexpressing PTTG.

Analysis of pituitary glands by electron microscopy revealed that especially gonadotrophs, but also thyrotrophs in bitransgenic {alpha}GSU.PTTGxRb+/– mice, exhibit morphological changes consistent with enhanced cellular activity, such as prominent Golgi apparatus and increased secretory granule number. The ultrastructure phenotype observed in {alpha}GSU.PTTG gonadotrophs is consistent with LH hypersecretion previously shown in these mice that results in hypertrophy of urogenital apparatus in males (19). In agreement with the pituitary volume profile observed with MRI, morphological changes by electron microscopy were more pronounced in bitransgenic {alpha}GSU.PTTGxRb+/– pituitaries compared with {alpha}GSU.PTTG pituitaries. Cytoplasmic glycogen accumulation observed in {alpha}GSU.PTTGxRb+/– gonadotrophs is unusual finding in pituitary gland, and the significance of this finding is not clear.

Confocal microscopy imaging shows that pituitary cells overexpressing PTTG exhibit clear nuclear changes. Altered chromatin pattern has been described in malignant cells, ranging from coarse and irregular distribution to peripheral margination of chromatin with central clearing of the nucleus (41). A model for nuclear organization suggests that progressive cellular differentiation relates to increasingly defined transcription patterns, morphologically translating in more compact chromatin, i.e. smaller nuclei and more evident repressed heterochromatin. Reversal of chromatin pattern toward totipotent embryonic phenotype, where nuclei are large and there is less repressed heterochromatin, may occur during meiosis and oncogenic transformation (42). Exact causes of changes of chromatin pattern in malignancy are not known, and possible explanations are: 1) chromatin relocation due to reactivation of repressed genes; 2) qualitative and quantitative changes in nuclear matrix protein; 3) acetylation of histones, disrupting nucleosome structure that leads to DNA relaxation for increased accessibility of transcription factors; 4) DNA aneuploidy; 5) increase in nuclear pore permeability; and 6) changes in attachment of lamins (proteins lining the inner nuclear membrane) to chromatin (41). Altered chromatin pattern caused by fixation method also needs to be considered; however, this is very unlikely in our pituitary samples because of the clear differences in nuclear pattern between controls (WT pituitary and eGPF-negative cells) and eGFP-positive cells. Macronuclei and chromatin redistribution were a distinct finding in cells expressing the {alpha}GSU-PTTG1-IRES-eGFP transgene. Interestingly, pituitary samples analyzed under confocal microscopy were derived from young mice (2 months old) and were nontumorous. As shown above, pituitary hyperplasia is already observed at this age, particularly in {alpha}GSU.PTTGxRb+/– mice. Confocal microscopy images show that hyperplastic pituitaries due to PTTG overexpression exhibit chromatin pattern alteration similar to the ones reported in fully malignant cells, suggesting that PTTG overexpression results in nuclear neoplastic phenotype. Nuclear vacuolization was found in a fraction of monotransgenic {alpha}GSU.PTTG and bitransgenic {alpha}GSU.PTTGxRb+/– pituitary cells, but the significance of this finding is not clear.

Increased pituitary hyperplasia in bitransgenic {alpha}GSU.PTTGxRb+/– mice directly correlated with higher frequency of tumors originating from PTTG-overexpressing cells, when compared with Rb+/– mice pituitaries, thus supporting a permissive role of PTTG in tumorigenesis. However, tumor cell proliferation as assessed by PCNA stain index was similar in anterior lobe tumors from both genotypes. This suggests that increased PTTG content results in higher tumor penetrance, but this does not lead to more aggressive tumors. Moreover, these tumors were negative for the antiapoptotic marker bcl-2 (data not shown), indicating that tumor growth does not rely on inhibition of apoptosis.

Several proposed mechanisms may explain the role of PTTG on tumor development. As a securin, PTTG has the critical role of regulating sister chromatid separation during mitosis. Abnormal PTTG levels cause asymmetric sister chromatid separation and aneuploidy (17). Genomic instability has been associated with cancer (43, 44, 45) and correlates with aggressive neoplasia (46, 47), including pituitary tumors (48). However, the cause and effect relation between aneuploidy and malignant tumors remains controversial (43). In disagreement with the hypothesis that genomic instability has a causal role on tumorigenesis, it has been shown that despite causing chromosome missegregation, Pttg inactivation does not predispose to tumor formation (18). Pttg–/– mouse embryo fibroblast metaphases exhibit quadriradial, triradial, and disrupted chromosomes, as well as premature centromere division. Nevertheless, Pttg knockout mice demonstrate tissue-specific hypoplasia and no increase in overall tumor prevalence (18). Therefore, aneuploidy caused by abnormal PTTG content is likely not wholly sufficient to initiate tumor formation.

In addition to its securin function, PTTG possesses transactivation ability (49). PTTG activates c-myc transcription (50) and inhibits expression of the cell cycle inhibitor p21 (13). PTTG binds to the c-myc promoter, and this may represent a mechanism for increased cell proliferation and transformation properties (15, 50) observed in cells with high PTTG content. Changes in pituitary PTTG content also inversely correlate with p21 expression, i.e. Pttg–/– pituitaries exhibit increased p21 and {alpha}GSU.PTTG pituitaries exhibit decreased p21 levels (13). PTTG inhibits p21 promoter activity, suggesting that p21 regulation by PTTG occurs at a transcriptional level (13). Decreased levels of the cyclin-dependent kinase inhibitor p21 in {alpha}GSU.PTTG pituitary glands may facilitate cell cycle progression that results in focal pituitary hyperplasia. In contrast, increased p21 levels, as seen in Pttg–/– mice, induces interphase cell cycle arrest and is likely a mechanism contributing to pituitary hypoplasia.

Another mechanism by which PTTG may facilitate tumor formation is by inducing growth factors. PTTG induces angiogenesis, as shown with conditioned media derived from PTTG-transfected cells (51). The angiogenic property of PTTG is mediated by fibroblast growth factor and vascular endothelial growth factor (51, 52), and PTTG, fibroblast growth factor, and vascular endothelial growth factor were all shown to be increased in pituitary tumors (53, 54).

Mechanisms that determine epigenetic PTTG regulation are currently being investigated, in particular the dynamic interaction between the Rb1 pathway and PTTG. PTTG perturbations are likely proximal events in the cascade of pituitary cell-transforming events ranging from hyperplasia to true adenoma formation. Understanding mechanisms for controlling pituitary plasticity may ultimately result in the ability to regulate both the rate of development and progression of pituitary tumors.


   Acknowledgments
 
We thank Dr. Run Yu for expertise in analyzing nuclear morphology and Dar-Yong Chen for performing mouse pituitary imaging.


   Footnotes
 
This work was supported by National Institutes of Health Grant CA 075979 (to S.M.) and by the Endocrine Fellows Foundation (to I.D.).

The authors have nothing to disclose.

First Published Online June 29, 2006

Abbreviations: eGFP, Enhanced green fluorescent protein; {alpha}GSU, {alpha}-subunit glycoprotein; MRI, magnetic resonance imaging; PRL, prolactin; PTTG, pituitary tumor transforming gene; RER, rough endoplasmic reticulum; RMANOVA, repeated measures ANOVA; TRITC, tetramethylrhodamine B isothiocyanate; WT, wild type.

Received April 25, 2006.

Accepted for publication June 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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