Early Multipotential Pituitary Focal Hyperplasia in the α-Subunit of Glycoprotein Hormone-Driven Pituitary Tumor-Transforming Gene Transgenic Mice (original) (raw)
Journal Article
,
1Departments of Medicine (R.A.A., I.T., E.M.B., S.-G.R., K.W., S.M.), Los Angeles, California 90048
Search for other works by this author on:
,
1Departments of Medicine (R.A.A., I.T., E.M.B., S.-G.R., K.W., S.M.), Los Angeles, California 90048
Search for other works by this author on:
,
1Departments of Medicine (R.A.A., I.T., E.M.B., S.-G.R., K.W., S.M.), Los Angeles, California 90048
Search for other works by this author on:
,
1Departments of Medicine (R.A.A., I.T., E.M.B., S.-G.R., K.W., S.M.), Los Angeles, California 90048
Search for other works by this author on:
,
2S. Mark Taper Imaging Center (D.-Y.C.), Cedars Sinai Research Institute, David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, California 90048
Search for other works by this author on:
,
1Departments of Medicine (R.A.A., I.T., E.M.B., S.-G.R., K.W., S.M.), Los Angeles, California 90048
Search for other works by this author on:
1Departments of Medicine (R.A.A., I.T., E.M.B., S.-G.R., K.W., S.M.), Los Angeles, California 90048
*Address all correspondence and requests for reprints to: Shlomo Melmed, M.D., Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Room 2015, Los Angeles, California 90048.
Search for other works by this author on:
Received:
11 October 2004
Accepted:
18 January 2005
Cite
Rula A. Abbud, Ichiro Takumi, Erin M Barker, Song-Guang Ren, Dar-Yong Chen, Kolja Wawrowsky, Shlomo Melmed, Early Multipotential Pituitary Focal Hyperplasia in the α-Subunit of Glycoprotein Hormone-Driven Pituitary Tumor-Transforming Gene Transgenic Mice, Molecular Endocrinology, Volume 19, Issue 5, 1 May 2005, Pages 1383–1391, https://doi.org/10.1210/me.2004-0403
Close
Navbar Search Filter Mobile Enter search term Search
Abstract
Pituitary tumor-transforming gene (PTTG), a securin protein isolated from pituitary tumor cell lines, is highly expressed in invasive tumors and exhibits characteristics of a transforming gene. To determine the role of PTTG in pituitary tumorigenesis, transgenic human PTTG1 was targeted to the mouse pituitary using the α-subunit of glycoprotein hormone. Males showed plurihormonal focal pituitary transgene expression with LH-, TSH-, and, unexpectedly, also GH-cell focal hyperplasia and adenoma, associated with increased serum LH, GH, testosterone, and/or IGF-I levels. MRI revealed both pituitary and prostate enlargement at 9–12 months. Urinary obstruction caused by prostatic hyperplasia and seminal vesicle hyperplasia, with renal tract inflammation, resulted in death by 10 months in some animals. Pituitary PTTG expression results in plurihormonal hyperplasia and hormone-secreting microadenomas with profound peripheral growth-stimulatory effects on the prostate and urinary tract. These results provide evidence for early pituitary plasticity, whereby PTTG overexpression results in a phenotype switch in early pituitary stem cells and promotes differentiated polyhormonal cell focal expansion.
PITUITARY CELL COMMITMENT and terminal differentiation follows a well-orchestrated temporal and spatial developmental cascade arising from multipotential cells (1–4). The mature gland comprises at least five highly differentiated hormone-secreting cell types.
Pituitary tumors develop due to intrinsic adenohypophysial cell alterations or altered growth factor availability (5, 6). Mutations in three genes have been linked to a familial predisposition to development of human pituitary tumors including multiple endocrine neoplasia type I (7), gsp (8), and PRKAR1a (9). Disrupted cell cycle regulatory genes, including Rb, p27, and p18, also result in murine pituitary tumor development, mainly in the intermediate lobe (10–12).
Pituitary tumor-transforming gene (PTTG), isolated by differential display from GH-secreting pituitary tumor cell lines (13), is expressed in actively proliferating normal tissue especially the testis and lymphopoietic system, and in several tumor types (13–20). PTTG is required for tissue self-renewal and _pttg_-null mice have hypoplastic testes, spleen, and pituitary glands (21). Male _pttg_-null mice also develop diabetes due to decreased pancreatic β-cell mass and proliferation (22).
PTTG acts as a securin protein essential for mitosis (23). During the cell cycle, a complex series of events ensures timely and equal separation of sister chromatids. During metaphase, sister chromatids are bound by cohesin, which is degraded by separin leading to chromatid separation at anaphase. PTTG binds separin and blocks chromatid separation at metaphase. Separase activation occurs upon PTTG degradation by the anaphase-promoting complex at the metaphase-anaphase transition. Thus, too much or too little PTTG results in chromosomal instability and aneuploidy (21, 24–26).
Several lines of evidence support the role of PTTG in tumorigenesis. Overexpressed PTTG induces cell aneuploidy (24), transforms NIH3T3 cells in vitro and in vivo (13), stimulates fibroblast growth factor production (6, 14, 20), and induces angiogenesis (5). Of genes associated with malignant cell behavior, PTTG was identified as one of nine genes comprising the “expression signature” for metastatic potential of solid tumors (27). Expression of a dominant-negative PTTG motif blocks experimental rat pituitary adenoma growth (28), whereas PTTG deletion protects Rb+/− mice from developing pituitary and thyroid tumors (29). These observations underscore the requirement of PTTG for pituitary tumorigenesis.
Pituitary cell differentiation and commitment follow a well-orchestrated temporal and spatial cascade arising from multipotential stem cells. Temporal and spatial expression of transcription factors and growth factors determine the specificity of hormone-secreting cell commitment. For example, Prop-1 determines specific GH, prolactin, and TSH expression (1), T-Pit is required for proopiomelanocortin gene expression (1, 30), and steroidogenic factor 1 is required for gonadotroph cell commitment (31). The dimeric glycoprotein hormones, FSH, LH, and TSH, are comprised of a common α-subunit [α-subunit of glycoprotein hormone (αGSU)] and a specific β-subunit. As αGSU is the earliest expressed pituitary hormone gene product (32), transgenic mice were generated with the αGSU promoter driving PTTG expression, to determine the impact of early pituitary PTTG expression. The results showing development of multihormonal tumors, by allowing hyperproliferation of early αGSU-expressing cells, lend credence to the presence of a multipotential early anterior pituitary stem cell.
Results
Targeted Pituitary Human (h) PTTG1 Expression in Transgenic Mice
To gain further insight into the in vivo requirement of PTTG for pituitary cell proliferation, transgenic mice expressing αGSU-directed hPTTG1 cDNA and enhanced green fluorescent protein (EGFP) were generated (Fig. 1A). Of four founder lines (three males and one female), one male founder failed to reproduce and developed intermediate lobe hyperplasia at 4 months of age, one was subfertile and gave rise to transgenic animals that failed to reproduce due to reproductive tract pathologies, and one sired transgenic offspring with a variety of phenotypes. Female transgenic mice were fertile and appeared healthy, and many offspring were obtained from the one female founder (Table 1). Female mice had enlarged pituitaries with elevated levels of serum IGF-I (αGSU.PTTG: 637 ± 135 vs. wild type: 250 ± 60 ng/ml; P < 0.05). A greater increase in pituitary size in response to pregnancy and lactation was also observed in transgenic females, as assessed by magnetic resonance imaging (MRI) (data not shown).
Fig. 1.
Generation of αGSU.PTTG Transgenic Mice A, αGSU.PTTG transgene construct consisting of the αGSU promoter driving expression of hPTTG1 and EGFP. IRES sequences allow for expression of both hPTTG1 and EGFP proteins. B, Mouse genotyping using Southern blot analysis. Tail DNA was digested with _Eco_RI and resolved on a 1.0% agarose gel. After transfer, the membrane was hybridized with a random-radiolabeled probe comprising the 1.2-kb _Xba_I fragment containing part of the bridging αGSU promoter and PTTG junction. Expected size of the transgenic fragment is 6.8 kb (arrow). P, Injected plasmid with vector digested with _Eco_RI as control; WT, wild type; TG, transgenic; IRES, internal ribosome entry site.
Table 1.
Breeding of Four αGSU.PTTG Transgenic Founders
Founder | Line 3 Male | Line 5 Male | Line 7 Male | Line 9 Female |
---|---|---|---|---|
F1 progeny (TG/WT) | 5 of 13 | None | 37 of 72 | 21 of 40 |
F2 progeny (TG/WT) | None | None | 35 of 83 | 26 of 49 |
F3 progeny (TG/WT) | None | None | 9 of 18 | 17 of 27 |
Founder | Line 3 Male | Line 5 Male | Line 7 Male | Line 9 Female |
---|---|---|---|---|
F1 progeny (TG/WT) | 5 of 13 | None | 37 of 72 | 21 of 40 |
F2 progeny (TG/WT) | None | None | 35 of 83 | 26 of 49 |
F3 progeny (TG/WT) | None | None | 9 of 18 | 17 of 27 |
TG, Transgenic; WT, wild type.
Table 1.
Breeding of Four αGSU.PTTG Transgenic Founders
Founder | Line 3 Male | Line 5 Male | Line 7 Male | Line 9 Female |
---|---|---|---|---|
F1 progeny (TG/WT) | 5 of 13 | None | 37 of 72 | 21 of 40 |
F2 progeny (TG/WT) | None | None | 35 of 83 | 26 of 49 |
F3 progeny (TG/WT) | None | None | 9 of 18 | 17 of 27 |
Founder | Line 3 Male | Line 5 Male | Line 7 Male | Line 9 Female |
---|---|---|---|---|
F1 progeny (TG/WT) | 5 of 13 | None | 37 of 72 | 21 of 40 |
F2 progeny (TG/WT) | None | None | 35 of 83 | 26 of 49 |
F3 progeny (TG/WT) | None | None | 9 of 18 | 17 of 27 |
TG, Transgenic; WT, wild type.
A summary of αGSU.PTTG phenotypes is shown in Table 2. There was a nonsignificant increase in body weight in αGSU.PTTG mice, and some male transgenic mice died prematurely at 8–12 months of age as a result of urinary tract obstruction and inflammation. MRI of male transgenic animals demonstrated larger and irregularly shaped pituitary glands than wild type (Fig. 2). Pituitary transgene expression was confirmed by demonstrating EGFP expression using scanning confocal microscopy. The EGFP signal was visualized on both the pituitary surface and up to 100 μm deep. Transgenic, but not wild-type, pituitary glands expressed EGFP in cell clusters assembled in two bilateral streaks in some animals (Fig. 3A). Rows of EGFP-positive cells juxtaposed to blood vessels were evident, and PTTG immunostaining showed focal PTTG expression (Fig. 3B). Focal PTTG expression was accompanied by loss of the reticulin network in some animals (Fig. 3E), indicating microadenoma rather than hyperplasia. Figure 3, C–F, depicts an example of a microadenoma coexpressing LH and PTTG, with vacuolizations.
Fig. 2.
Evidence of Transgenic Pituitary Enlargement and Irregular Shape on MRI Sagittal (A) and coronal (B) MRI images of one wild-type and two transgenic (αGSU.PTTG) mice. C, Scattergram depicting pituitary size in total pixels obtained by adding the pituitary area obtained from consecutive sagittal images. WT, Wild type.
Fig. 3.
Pituitary PTTG Expression in αGSU.PTTG Mice Results in Focal Hyperplasia and Adenoma A, Scanning confocal images showing EGFP signal (green) appearing as two bilateral streaks in αGSU.PTTG pituitary. B, Focal PTTG expression in a representative αGSU.PTTG mouse pituitary sections (5 μm) immunostained for PTTG (brown) and counterstained with hematoxylin (blue). Magnification: ×100. C–F, Representative pituitary sections from αGSU.PTTG animal that developed a highly vacuolized (arrows) PTTG (panel D, red) and LH-immunostained (panel F, red) tumor, as evidenced by the disrupted reticulin network (panel E, black fibers). Magnification: C, ×40; D, ×400; E, ×100; F: ×200.
Table 2.
Summary of Observed αGSU.PTTG Phenotypes
| | Wild Type | αGSU.PTTG | | | --------------------------------------- | ----------------------------------------------- | ----------------------------------------------------- | | Weight (g) | 44.5 ± 2.6 | 50.7 ± 2.2 (all lines) | | Premature death (8–12 months) | 0 of 10 | 6 of 28 (all lines) | | Pituitary MRI (pixels) | 191 ± 5.5 | 242.7 ± 13.1a | | | (n = 5) | (line 3: n = 5; line 7: n = 5) | | | Serum Levels (line no. 7): | | | | FSH (ng/ml) | 52 ± 7 | 45 ± 3 | | T4 (ng/dl) | 0.3 ± 0.04 | 0.3 ± 0.02 | | Testosterone (ng/dl) | 258 ± 94 | 693 ± 153a | | IGF-I (ng/ml) | 377 ± 56 | 496 ± 42a | | GH (ng/ml) | 1.1 ± 0.7 | 15.2 ± 7.1 | | LH (ng/ml) | 0.5 ± 0.3 | 2 ± 0.8 | | Pathology: (lines 3, 7, and 9, F0 & F1) | | | | Prostate | | Inflammation: 3 of 8 | | | | Hyperplasia: 4 of 8 | | | | | Tumor: 1 of 8 | | | Seminal vesicles | | Enlarged: 24 of 28 | | | | Hemorrhagic: 11 of 28 | | | Urinary bladder | | Inflammatory bladder neck obstruction: 9 of 28 | | Pituitary pathology | | Vacuolizations: 11 of 16 | | | | Focal PTTG expression: 9 of 16 | | | | | Abnormal pituitary gland in all lines: 29 of 38 | |
| | Wild Type | αGSU.PTTG | | | --------------------------------------- | ----------------------------------------------- | ----------------------------------------------------- | | Weight (g) | 44.5 ± 2.6 | 50.7 ± 2.2 (all lines) | | Premature death (8–12 months) | 0 of 10 | 6 of 28 (all lines) | | Pituitary MRI (pixels) | 191 ± 5.5 | 242.7 ± 13.1a | | | (n = 5) | (line 3: n = 5; line 7: n = 5) | | | Serum Levels (line no. 7): | | | | FSH (ng/ml) | 52 ± 7 | 45 ± 3 | | T4 (ng/dl) | 0.3 ± 0.04 | 0.3 ± 0.02 | | Testosterone (ng/dl) | 258 ± 94 | 693 ± 153a | | IGF-I (ng/ml) | 377 ± 56 | 496 ± 42a | | GH (ng/ml) | 1.1 ± 0.7 | 15.2 ± 7.1 | | LH (ng/ml) | 0.5 ± 0.3 | 2 ± 0.8 | | Pathology: (lines 3, 7, and 9, F0 & F1) | | | | Prostate | | Inflammation: 3 of 8 | | | | Hyperplasia: 4 of 8 | | | | | Tumor: 1 of 8 | | | Seminal vesicles | | Enlarged: 24 of 28 | | | | Hemorrhagic: 11 of 28 | | | Urinary bladder | | Inflammatory bladder neck obstruction: 9 of 28 | | Pituitary pathology | | Vacuolizations: 11 of 16 | | | | Focal PTTG expression: 9 of 16 | | | | | Abnormal pituitary gland in all lines: 29 of 38 | |
Table 2.
Summary of Observed αGSU.PTTG Phenotypes
| | Wild Type | αGSU.PTTG | | | --------------------------------------- | ----------------------------------------------- | ----------------------------------------------------- | | Weight (g) | 44.5 ± 2.6 | 50.7 ± 2.2 (all lines) | | Premature death (8–12 months) | 0 of 10 | 6 of 28 (all lines) | | Pituitary MRI (pixels) | 191 ± 5.5 | 242.7 ± 13.1a | | | (n = 5) | (line 3: n = 5; line 7: n = 5) | | | Serum Levels (line no. 7): | | | | FSH (ng/ml) | 52 ± 7 | 45 ± 3 | | T4 (ng/dl) | 0.3 ± 0.04 | 0.3 ± 0.02 | | Testosterone (ng/dl) | 258 ± 94 | 693 ± 153a | | IGF-I (ng/ml) | 377 ± 56 | 496 ± 42a | | GH (ng/ml) | 1.1 ± 0.7 | 15.2 ± 7.1 | | LH (ng/ml) | 0.5 ± 0.3 | 2 ± 0.8 | | Pathology: (lines 3, 7, and 9, F0 & F1) | | | | Prostate | | Inflammation: 3 of 8 | | | | Hyperplasia: 4 of 8 | | | | | Tumor: 1 of 8 | | | Seminal vesicles | | Enlarged: 24 of 28 | | | | Hemorrhagic: 11 of 28 | | | Urinary bladder | | Inflammatory bladder neck obstruction: 9 of 28 | | Pituitary pathology | | Vacuolizations: 11 of 16 | | | | Focal PTTG expression: 9 of 16 | | | | | Abnormal pituitary gland in all lines: 29 of 38 | |
| | Wild Type | αGSU.PTTG | | | --------------------------------------- | ----------------------------------------------- | ----------------------------------------------------- | | Weight (g) | 44.5 ± 2.6 | 50.7 ± 2.2 (all lines) | | Premature death (8–12 months) | 0 of 10 | 6 of 28 (all lines) | | Pituitary MRI (pixels) | 191 ± 5.5 | 242.7 ± 13.1a | | | (n = 5) | (line 3: n = 5; line 7: n = 5) | | | Serum Levels (line no. 7): | | | | FSH (ng/ml) | 52 ± 7 | 45 ± 3 | | T4 (ng/dl) | 0.3 ± 0.04 | 0.3 ± 0.02 | | Testosterone (ng/dl) | 258 ± 94 | 693 ± 153a | | IGF-I (ng/ml) | 377 ± 56 | 496 ± 42a | | GH (ng/ml) | 1.1 ± 0.7 | 15.2 ± 7.1 | | LH (ng/ml) | 0.5 ± 0.3 | 2 ± 0.8 | | Pathology: (lines 3, 7, and 9, F0 & F1) | | | | Prostate | | Inflammation: 3 of 8 | | | | Hyperplasia: 4 of 8 | | | | | Tumor: 1 of 8 | | | Seminal vesicles | | Enlarged: 24 of 28 | | | | Hemorrhagic: 11 of 28 | | | Urinary bladder | | Inflammatory bladder neck obstruction: 9 of 28 | | Pituitary pathology | | Vacuolizations: 11 of 16 | | | | Focal PTTG expression: 9 of 16 | | | | | Abnormal pituitary gland in all lines: 29 of 38 | |
Because the mouse αGSU promoter targets transgene expression to both gonadotropes and thyrotropes (32), double-label immunocytochemistry was performed to determine hormonal PTTG coexpression. Control pituitary glands derived from wild-type littermates did not exhibit appreciable PTTG immunofluorescence, but transgenic pituitaries expressed PTTG immunoreactivity in most, but not all, αGSU-expressing cells. Figure 4, A and B show αGSU staining using fluorescein-labeled antibodies (green) and PTTG staining with Texas Red (red). Double-labeled cells are yellow. PTTG was expressed in some, but not all, αGSU cells as well as other cell types. Because LH and TSH are coexpressed with αGSU, pituitary sections were costained for LH (Fig. 4D), TSH (Fig. 4E), and PTTG. PTTG was expressed in both LH and TSH. Surprisingly, non-αGSU cells that expressed PTTG also costained for GH (Fig. 4C), and some GH cells expressed αGSU (Fig. 4F). Focal expansion of αGSU, LH, and/or GH cells was observed in some animals (Fig. 5), whereas expression of pituitary ACTH and prolactin was normal (data not shown).
Fig. 4.
Polyhormonal PTTG Coexpression in αGSU.PTTG Mouse Pituitary A and B, Double-label immunostaining of a transgenic mouse pituitary for PTTG (red) and αGSU (green). Overlay of PTTG and αGSU images with Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) reveals double-labeled cells in yellow. Magnification: ×1000. C, Double-label immunostaining for PTTG (brown) and GH (pink) of transgenic mouse pituitary, showing costaining of some GH and PTTG cells (arrows) with ribbon-like appearance of adenomatous tumor cells. Magnification: ×1000. D, Double-label immunocytochemistry for LH (green) and PTTG (red) with costained cells appearing in yellow. Magnification: ×200. E, Coexpression of some, but not all, TSH- (pink) and PTTG-expressing (brown) cells. Arrow indicates double-labeled cells and arrowhead points to a TSH cell not expressing PTTG. Note the ribbon-like pattern of PTTG expression. Magnification: ×200. F, Representative pituitary section from an animal with elevated IGF-I levels costained for αGSU (green) and GH (red). Focal hyperplasia of GH-expressing cells was observed with coexpression of αGSU with GH in some cells (arrows). Asterisk depicts αGSU cell that did not express GH. Magnification: ×400.
Fig. 5.
Development of Plurihormonal Focal Adenomas in αGSU.PTTG Mice A–H, Pituitary sections from a representative animal with PTTG-expressing cells organized in a ribbon-like or bead-on-a-string-like pattern. Sections were costained with PTTG (brown, panels E–H) and one of the following: TSH (red, panel A), LH (red, panel B), αGSU (red, panel C), and GH (red, panel D), respectively. Magnification: ×1000. I–J, Representative sections from an animal with LH-producing (red) adenoma that did not immunostain for GH (green). Magnification: I, ×200; J, ×400.
Pathological findings in transgenic pituitaries consisted of microscopic focal expansion of hormone-specific cells with ribbon-like pattern of tumor cells and vacuolizations. Exclusion of other immunoreactive hormones from these areas was indicative of cell specificity. Loss of reticulin network was also a hallmark of adenomatous change. Large macroscopic adenomas were not observed, likely because animals died early from renal tract obstruction.
Shown in Fig. 5 are two representative male transgenic pituitaries; one with a plurihormonal focal adenoma consisting of ribbon-like PTTG-expressing cells that costained for αGSU, LH, TSH, and GH (Fig. 5, A–H), whereas the other exhibited focal expansion of LH cells alone (Fig. 5, I and J). Thus, evidence for focal LH- or GH- or TSH-cell adenoma formation in transgenic male pituitaries included the ribbon-like pattern of adenoma cells and reticulin loss. Presence of extensive pituitary vacuolization further supported the adenomatous nature of the focal cell expansions (33, 34).
Hormone Levels
Table 2 depicts serum hormone levels observed in wild-type and transgenic mice. FSH, TSH, and T4 levels were not different in male transgenic from wild-type mice (Table 2). LH and GH levels were elevated in some, but not all, transgenic animals (Table 2 and Fig. 6, A and B). However, mean serum testosterone and IGF-I levels were higher in transgenic than in wild-type mice (Table 2 and Fig. 6, C and D), as were testicular testosterone levels (data not shown).
Fig. 6.
Hypersecretion of LH, GH, Testosterone, and/or IGF-I in αGSU.PTTG Mice Serum hormone levels were measured by RIA and values were normalized to those obtained from wild-type littermates. Individual data points are plotted for both wild-type and transgenic (αGSU.PTTG) mice. WT, Wild type.
Male Genitourinary Tract Pathology
The most striking phenotype observed in αGSU.PTTG male transgenic mice was that of urinary tract obstruction secondary to prostate hyperplasia evident at 8–12 months of age. The urinary bladder was enlarged with wall thickening and filled with urine containing inflammatory cells and white deposits. Seminal vesicles were also enlarged (Fig. 7), but testicular weight was unchanged. Seminal vesicle histology showed fibromuscular stromal thickening with signs of inflammation or adenoma in some animals (Fig. 7). Histological examination of the prostate revealed focal micropapillary hyperplasia with dilated ducts, cell atypia, and prostate intraepithelial neoplasia. It is characterized by multifocal proliferative regions of atypical epithelial cells in multiple ductules with cribriform and/or tufting growth patterns with progressive nuclear atypia. These patterns appear as bridges of epithelial cells within each ductule. In some animals that died prematurely, stromal hyperplasia, and inflammatory prostatitis were observed (Fig. 8).
Fig. 7.
Seminal Vesicle Pathology in αGSU.PTTG Mice Shown are representative seminal vesicles (outlined in white) derived from wild-type (A) and αGSU.PTTG transgenic (B) animals. Transgenic animals had enlarged seminal vesicles with increased lumen size. C, Representative seminal vesicle section (5 μm) stained with hematoxylin and eosin showing thickening of the fibromuscular stroma (*), signs of inflammation (black arrowhead), and adenoma (white arrow).
Fig. 8.
Prostate Pathology in αGSU.PTTG Mice A and B, MRI of wild-type and transgenic (αGSU.PTTG) mice showing enlargement of the ventral prostate (arrows) in transgenic as compared with wild type. Prostate enlargement leads to narrowing of the urethral opening (*). C–E, Hematoxylin and eosin-stained sections from wild-type and transgenic (αGSU.PTTG) prostate glands, with transgenic prostate showing intraepithelial neoplasia with multifocal proliferative regions of atypical epithelial cells forming bridges (arrow) with cribriform and tufting growth patterns. E, Evidence for inflammatory prostatitis (arrows) in αGSU.PTTG mouse prostate section stained with hematoxylin and eosin. WT, Wild type.
Discussion
Targeted expression of PTTG using the mouse αGSU promoter results in focal PTTG expression in LH- and GH-producing cells ranging from hyperplasia to frank adenoma development. The finding of GH cell hyperplasia was surprising because the αGSU promoter has not been shown to drive expression to mature somatotrope cells. Increased GH and LH result in elevated IGF-I and testosterone levels, respectively, resulting in marked prostate and seminal vesicle neoplasia. Prostate hyperplasia results in bladder obstruction, kidney reflex, and inflammation. Some phenotypic features of αGSU.PTTG male mice are reminiscent of previously reported defects in mice that overexpress human chorionic gonadotropin (35, 36), suggesting a role for LH overexpression in the pathogenesis of prostate hyperplasia. These observations suggest that PTTG overexpression in the developing pituitary targets early pituitary multipotential cells or, alternatively, may influence neighboring non-αGSU-expressing cells by a paracrine mechanism.
How is transgene expression in GH cells explained? The mouse αGSU promoter targets PTTG expression to pituitary stem cells early in development, with the potential to give rise to all pituitary hormone cell types. Nevertheless, mature somatotrope cells rarely express αGSU. However, Camper and co-workers (37) labeled early embryonic cells with β-galactosidase and showed that all pituitary cells, including GH cells, appear to originate from αGSU progenitor cells. Nevertheless, why PTTG is not suppressed in GH cells remains to be determined. Some GH cells express αGSU modestly (38), as do GH-secreting tumor cells (39, 40), supporting the hypothesis for a common plastic pituitary precursor lineage. Overexpressed thyrotrope PTTG may have resulted in thyrotrope hyperplasia and transdifferentiation of these cells into somatotropes, because these cells share the common Pit-1 lineage. Transdifferentiation of GH-secreting from TSH-secreting cells has been reported in states of hypothyroidism leading to TSH hyperplasia (41). Gonadotrope PTTG overexpression may also result in paracrine regulation of GH cell proliferation. For example, female transgenic mice that hypersecrete LH also exhibit elevated serum GH levels (42, 43).
Recent observations have suggested that a subpopulation of embryonic pituitary cells may coexpress two or more hormone mRNAs. αGSU with GH and/or prolactin expression are the earliest coexpressed hormones occurring at embryonic d 16. Age-related changes in combined single-cell hormone coexpression did not correspond with those observed for single hormone expression, suggesting a unique response of coexpressing adenohypophysial cells to developmental signals (38). The results shown here, whereby αGSU-driven PTTG gives rise to adenomas of both glycoprotein hormones as well as GH-secreting cells, provide further evidence for the coexpression of αGSU in embryonic somatotropes.
Prostate, seminal vesicle, and urinary tract pathology is attributed to increased pituitary secretion of both LH and GH, resulting in elevated testosterone and IGF-I levels, respectively. Although high levels of all four hormones were rarely observed in the same animal, most αGSU.PTTG male mice had abnormally elevated levels of at least one of these hormones. Not surprisingly, these animals developed prostate pathology as both testosterone (44) and IGF-I (45–49) have been linked to prostate hyperplasia and tumors. As the animals aged, prostate enlargement resulted in urinary tract obstruction leading to urinary bladder enlargement, inflammation, and even pyelonephritis in some animals.
These studies suggest that early overexpressed PTTG results in proliferation of multihormonal pituitary cells, underscoring the role of PTTG in pituitary cell proliferation and adenoma formation, and also point to the presence of a multipotential early pituitary stem cell expressing αGSU. Cell vacuolization is a hallmark of acidophilic stem cell adenoma. The large clear vacuoles observed in acidophilic stem cell adenoma are due to mitochondrial accumulation that can be seen as a giant mitochondrion under electron microscopy, which is indicative of oncocytic change (34). Previous studies have shown that PTTG abundance is increased in several tumor types, and its overexpression results in cell transformation. Our results validate that in vivo overexpression of PTTG itself induces abnormal pituitary cell proliferation and adenomas. Potential mechanisms include induction of aneuploidy as a result of dysregulation of sister chromatid separation (24, 50), regulation of other cell cycle proteins, including p53 (51, 52), or transactivation with other transcription factors involved in pituitary proliferation (19, 53).
Materials and Methods
DNA Constructs
The hPTTG1-IRES2-EGFP plasmid was constructed by replacement of PCMV by the 4.6 kb (_Kpn_I-_Hin_dIII) fragment of the rat αGSU promoter region [generously provided by Dr. E. C. Ridgway (32)], and by insertion of a 654-bp (Xho I-_Mlu_I) fragment containing the full-length hPTTG1 cDNA into the polylinker region of the pIRES2-EGFP vector (CLONTECH Laboratories, Inc., Palo Alto, CA) with the following restriction sites replaced by linkers: _Ase_I by _Sac_II and _Afl_II by _Sfi_I, respectively (Fig. 1A).
Transgenic Mice
hPTTG1-IRES2-EGFP was digested with _Kpn_I and _Afl_II, the transgene was microinjected into B6C3-fertilized mouse pronuclei, and injected eggs were transplanted to pseudopregnant foster mothers at the University of California Los Angeles Transgenic Core Facility. For genotyping, either Southern blots or PCR using EGFP primers (AGAACGGCATCAAGGTGAAC and CAGAAGAACGGCATCAAGGT) were performed. All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee. Briefly, mice were housed in microisolator cages and cubicles in a room with 12-h light, 12-h dark cycle. Animals were euthanized using CO2 chambers, and blood was withdrawn directly from the heart. Pituitary glands were collected and fixed in 2% paraformaldehyde for 2 h whereas remaining organs were fixed in formalin.
Southern Blots
Tail genomic DNA samples were digested with _Eco_RI and resolved on 1% agarose gel. After DNA transfer to Hybond-n + (Amersham Pharmacia Biotech, Arlington Heights, IL) membrane, it was hybridized with a radiolabeled probe comprising a 1.2-kb _Xba_I fragment containing the junction of the αGSU promoter and hPTTG1 (Fig. 1B). Upon exposure of the membrane to film, a 6.8-kb band appears in transgenic samples. Injected plasmid DNA digested with _Eco_RI was used as control.
Confocal Microscopy
Scanning confocal images were obtained using a confocal microscope TCS-SP confocal scanner (Leica Microsystems, Mannheim, Germany). Images were taken with a ×10, 0.3 N.A. Plan Fluotar. The objective provides a scan field of 1 × 1 mm. Because the pituitary is significantly larger, we performed a tiled scan. The complete imaging field was divided into three × four quadrants. Each quadrant was scanned separately as a 200-μm deep stack with 7-μm spaced optical sections along the z-axis. The sample was positioned by a Scan motorized stage (Märzhäuser, Wertzlar, Germany), maximum intensity projection was calculated for each stack, and aligned projections were assembled into the final figure. The spectrophotometer was set to optimal EGFP detection to a wide setting of 500–590 nm. Pinhole was set to 1.5 Airy units to provide for depth penetration and efficient light collection.
Histology
Tissue sectioning was performed by the Department of Pathology at Cedars Sinai Medical Center. For the pituitary, 4-μm sections of paraformaldehyde- (2% in PBS for 2 h) fixed and paraffin-embedded tissue were obtained and stained for hematoxylin and eosin or reticulin silver stain. Double-labeled immunocytochemistry was performed using a previously optimized protocol (42, 43) or the EnVision kit (DAKO Cytomation, Inc., Carpinteria, CA). Antibodies used were: rabbit polyclonal anti-PTTG-1 (Zymed Laboratories Inc., South San Francisco, CA, 1:2000), Rabbit antihuman ACTH [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), 1:200], rabbit antirat GH (NIDDK, 1:200), guinea pig antirat α-subunit (NIDDK, 1:200), guinea pig antirat β-LH (NIDDK, 1:200), guinea pig antirat prolactin (NIDDK, 1:200), rabbit antirat TSHβ (NIDDK, 1:1000). Secondary antibodies included fluorescein thiocyanate-labeled antirabbit, Cy3-labeled antirabbit, Rhodamine-labeled antirabbit, and fluorescein thiocyanate-labeled antiguinea pig. When the Envision kit was used, secondary antibodies and chromagens were used according to manufacturer specification. For PTTG immunostaining, an antigen-retrieval step was performed before incubation with primary antibody. Slides were counterstained with 4′,6-diamidino-2-phenylindole or hematoxylin to visualize nuclei.
Ria
Serum IGF-I was measured using the rat IGF-I RIA kit (DSL, Inc., Webster, TX). A solid-phase RIA for measurement of total testosterone (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA) in diethyl ether extracted serum samples. The DPC Coat-A-Count total T4 assay was also used. For GH measurements, a rat GH RIA was used as previously described (54). LH and FSH levels were assayed by the University of Virginia Center for Cellular and Molecular studies in Reproduction.
Mri
We used a clinical whole-body 1.5 Tesla MRI system (Siemens Visions, Madison, WI) to image the mouse pituitary. Procedures were performed during nonclinical hours between 2100 h and 0700 h, and equipment was disinfected before and after animal imaging. Animals were anesthetized with avertin and imaged using a small solenoidal receiver coil. We obtained T1-weighted spin echo images (repetition time, 400 msec; echo time, 14 msec; number of signal averages, 4; imaging time, 6 min, 53 sec; slice thickness, 1 mm; in-plane resolution, 195 μm) in the coronal and sagittal imaging planes. Pituitary volume was determined by multiplying pixel volume by the number of pixels within the pituitary gland as defined by a region of interest that was manually drawn on magnified sagittal images using the MRI system console.
Statistical Analysis
Because most comparisons were made between wild-type and transgenic littermates, an unpaired Student’s t test was used to determine statistical significances, which are determined at P < 0.05.
Acknowledgments
This work was supported by National Institutes of Health (NIH) Grant CA 075979 (to S.M.), the Yoshida Foundation, and the Janameg (to I.T.). The Center for Cellular and Molecular Studies in Reproduction at the University of Virginia was supported by NIH Grant U54-HD28–934.
The authors thank Dr. Dan Haisenleder and Ms. Aleisha Schoenfelder for their assistance with RIAs through the Center for Cellular and Molecular Studies in Reproduction at the University of Virginia. We also thank Dr. Toni Prezant, Dr. Jonathan Said, and Ms. Liz Lantsey for their helpful expertise.
Abbreviations:
- EGFP,
Enhanced green fluorescent protein; - αGSU,
α-subunit of glycoprotein hormone; - MRI,
magnetic resonance imaging; - PTTG,
pituitary tumor-transforming gene.
References
1
Burgess
R
,
Lunyak
V
,
Rosenfeld
M
2002
Signaling and transcriptional control of pituitary development.
Curr Opin Genet Dev
12
:
534
–
539
2
Watkins-Chow
DE
,
Camper
SA
1998
How many homeobox genes does it take to make a pituitary gland?
Trends Genet
14
:
284
–
290
3
Drouin
J
,
Lamolet
B
,
Lamonerie
T
,
Lanctot
C
,
Tremblay
JJ
1998
The PTX family of homeodomain transcription factors during pituitary developments.
Mol Cell Endocrinol
140
:
31
–
36
4
Alarid
ET
,
Holley
S
,
Hayakawa
M
,
Mellon
PL
1998
Discrete stages of anterior pituitary differentiation recapitulated in immortalized cell lines.
Mol Cell Endocrinol
140
:
25
–
30
5
Melmed
S
2003
Mechanisms for pituitary tumorigenesis: the plastic pituitary.
J Clin Invest
112
:
1603
–
1618
6
Heaney
AP
,
Melmed
S
2004
Molecular targets in pituitary tumours.
Nat Rev Cancer
4
:
285
–
295
7
Marx
SJ
,
Agarwal
SK
,
Kester
MB
,
Heppner
C
,
Kim
YS
,
Skarulis
MC
,
James
LA
,
Goldsmith
PK
,
Saggar
SK
,
Park
SY
,
Spiegel
AM
,
Burns
AL
,
Debelenko
LV
,
Zhuang
Z
,
Lubensky
IA
,
Liotta
LA
,
Emmert-Buck
MR
,
Guru
SC
,
Manickam
P
,
Crabtree
J
,
Erdos
MR
,
Collins
FS
,
Chandrasekharappa
SC
1999
Multiple endocrine neoplasia type 1: clinical and genetic features of the hereditary endocrine neoplasias.
Recent Prog Horm Res
54
:
397
–
438
8
Spada
A
,
Vallar
L
,
Faglia
G
1993
G-proteins and hormonal signalling in human pituitary tumors: genetic mutations and functional alterations.
Front Neuroendocrinol
14
:
214
–
232
9
Stratakis
CA
,
Matyakhina
L
,
Courkoutsakis
N
,
Patronas
N
,
Voutetakis
A
,
Stergiopoulos
S
,
Bossis
I
,
Carney
JA
2004
Pathology and molecular genetics of the pituitary gland in patients with the ‘complex of spotty skin pigmentation, myxomas, endocrine overactivity and schwannomas’ (Carney complex).
Front Horm Res
32
:
253
–
264
10
Franklin
DS
,
Godfrey
VL
,
Lee
H
,
Kovalev
GI
,
Schoonhoven
R
,
Chen-Kiang
S
,
Su
L
,
Xiong
Y
1998
CDK inhibitors p18(INK4c) and p27(Kip1) mediate two separate pathways to collaboratively suppress pituitary tumorigenesis.
Genes Dev
12
:
2899
–
2911
11
Park
MS
,
Rosai
J
,
Nguyen
HT
,
Capodieci
P
,
Cordon-Cardo
C
,
Koff
A
1999
p27 and Rb are on overlapping pathways suppressing tumorigenesis in mice.
Proc Natl Acad Sci USA
96
:
6382
–
6387
12
Yamasaki
L
,
Bronson
R
,
Williams
BO
,
Dyson
NJ
,
Harlow
E
,
Jacks
T
1998
Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1(+/−)mice.
Nat Genet
18
:
360
–
364
13
Pei
L
,
Melmed
S
1997
Isolation and characterization of a pituitary tumor-transforming gene (PTTG).
Mol Endocrinol
11
:
433
–
441
14
McCabe
CJ
,
Khaira
JS
,
Boelaert
K
,
Heaney
AP
,
Tannahill
LA
,
Hussain
S
,
Mitchell
R
,
Olliff
J
,
Sheppard
MC
,
Franklyn
JA
,
Gittoes
NJ
2003
Expression of pituitary tumour transforming gene (PTTG) and fibroblast growth factor-2 (FGF-2) in human pituitary adenomas: relationships to clinical tumour behaviour.
Clin Endocrinol (Oxf)
58
:
141
–
150
15
Saez
C
,
Pereda
T
,
Borrero
JJ
,
Espina
A
,
Romero
F
,
Tortolero
M
,
Pintor-Toro
JA
,
Segura
DI
,
Japon
MA
2002
Expression of hpttg proto-oncogene in lymphoid neoplasias.
Oncogene
21
:
8173
–
8177
16
Shibata
Y
,
Haruki
N
,
Kuwabara
Y
,
Nishiwaki
T
,
Kato
J
,
Shinoda
N
,
Sato
A
,
Kimura
M
,
Koyama
H
,
Toyama
T
,
Ishiguro
H
,
Kudo
J
,
Terashita
Y
,
Konishi
S
,
Fujii
Y
2002
Expression of PTTG (pituitary tumor transforming gene) in esophageal cancer.
Jpn J Clin Oncol
32
:
233
–
237
17
Saez
C
,
Japon
MA
,
Ramos-Morales
F
,
Romero
F
,
Segura
DI
,
Tortolero
M
,
Pintor-Toro
JA
1999
hpttg Is over-expressed in pituitary adenomas and other primary epithelial neoplasias.
Oncogene
18
:
5473
–
5476
18
Dominguez
A
,
Ramos-Morales
F
,
Romero
F
,
Rios
RM
,
Dreyfus
F
,
Tortolero
M
,
Pintor-Toro
JA
1998
hpttg, A human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG.
Oncogene
17
:
2187
–
2193
19
Yu
R
,
Melmed
S
2004
Pituitary tumor transforming gene: an update.
Front Horm Res
32
:
175
–
185
20
Heaney
AP
,
Horwitz
GA
,
Wang
Z
,
Singson
R
,
Melmed
S
1999
Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis.
Nat Med
5
:
1317
–
1321
21
Wang
Z
,
Yu
R
,
Melmed
S
2001
Mice lacking pituitary tumor transforming gene show testicular and splenic hypoplasia, thymic hyperplasia, thrombocytopenia, aberrant cell cycle progression, and premature centromere division.
Mol Endocrinol
15
:
1870
–
1879
22
Wang
Z
,
Moro
E
,
Kovacs
K
,
Yu
R
,
Melmed
S
2003
Pituitary tumor transforming gene-null male mice exhibit impaired pancreatic β cell proliferation and diabetes.
Proc Natl Acad Sci USA
100
:
3428
–
3432
23
Orr-Weaver
TL
1999
Perspectives: cell cycle. The difficulty in separating sisters.
Science
285
:
344
–
345
24
Yu
R
,
Lu
W
,
Chen
J
,
McCabe
CJ
,
Melmed
S
2003
Overexpressed pituitary tumor-transforming gene causes aneuploidy in live human cells.
Endocrinology
144
:
4991
–
4998
25
Zou
H
,
McGarry
TJ
,
Bernal
T
,
Kirschner
MW
1999
Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis.
Science
285
:
418
–
422
26
Waizenegger
I
,
Gimenez-Abian
JF
,
Wernic
D
,
Peters
JM
2002
Regulation of human separase by securin binding and autocleavage.
Curr Biol
12
:
1368
–
1378
27
Ramaswamy
S
,
Ross
KN
,
Lander
ES
,
Golub
TR
2003
A molecular signature of metastasis in primary solid tumors.
Nat Genet
33
:
49
–
54
28
Horwitz
GA
,
Miklovsky
I
,
Heaney
AP
,
Ren
SG
,
Melmed
S
2003
Human pituitary tumor-transforming gene (PTTG1) motif suppresses prolactin expression.
Mol Endocrinol
17
:
600
–
609
29
Chesnokova
V
,
Castro
A-V
,
Kovacs
K
,
Melmed
S
,
Pttg is required for pituitary tumor development in Rb-deficient mice.
Program of the 86th Annual Meeting of The Endocrine Society
,
New Orleans, LA
,
2004
, p
127
(Abstract OR37-2)
30
Pulichino
AM
,
Vallette-Kasic
S
,
Couture
C
,
Gauthier
Y
,
Brue
T
,
David
M
,
Malpuech
G
,
Deal
C
, Van
Vliet
G
, De
Vroede
M
,
Riepe
FG
,
Partsch
CJ
,
Sippell
WG
,
Berberoglu
M
,
Atasay
B
,
Drouin
J
2003
Human and mouse TPIT gene mutations cause early onset pituitary ACTH deficiency.
Genes Dev
17
:
711
–
716
31
Bakke
M
,
Zhao
L
,
Parker
KL
2001
Approaches to define the role of SF-1 at different levels of the hypothalamic-pituitary-steroidogenic organ axis.
Mol Cell Endocrinol
179
:
33
–
37
32
Kendall
SK
,
Gordon
DF
,
Birkmeier
TS
,
Petrey
D
,
Sarapura
VD
,
O’Shea
KS
,
Wood
WM
,
Lloyd
RV
,
Ridgway
EC
,
Camper
SA
1994
Enhancer-mediated high level expression of mouse pituitary glycoprotein hormone α-subunit transgene in thyrotropes, gonadotropes, and developing pituitary gland.
Mol Endocrinol
8
:
1420
–
1433
33
Wenig
B
,
Heffess
C
,
Adair
C
1997
Atlas of endocrine pathology.
1st ed.
Philadelphia
:
W.B. Saunders Co.
34
Asa
SL
1998
Atlas of tumor pathology: tumors of the pituitary.
3rd series ed.
Washington, DC
:
Armed Forces Institute of Pathology
35
Matzuk
MM
,
DeMayo
FJ
,
Hadsell
LA
,
Kumar
TR
2003
Overexpression of human chorionic gonadotropin causes multiple reproductive defects in transgenic mice.
Biol Reprod
69
:
338
–
346
36
Rulli
SB
,
Ahtiainen
P
,
Makela
S
,
Toppari
J
,
Poutanen
M
,
Huhtaniemi
I
2003
Elevated steroidogenesis, defective reproductive organs, and infertility in transgenic male mice overexpressing human chorionic gonadotropin.
Endocrinology
144
:
4980
–
4990
37
Cushman
LJ
,
Burrows
HL
,
Seasholtz
AF
,
Lewandoski
M
,
Muzyczka
N
,
Camper
SA
2000
Cre-mediated recombination in the pituitary gland.
Genesis
28
:
167
–
174
38
Seuntjens
E
,
Hauspie
A
,
Vankelecom
H
,
Denef
C
2002
Ontogeny of plurihormonal cells in the anterior pituitary of the mouse, as studied by means of hormone mRNA detection in single cells.
J Neuroendocrinol
14
:
611
–
619
39
White
MC
,
Newland
P
,
Daniels
M
,
Turner
SJ
,
Mathias
D
,
Teasdale
G
,
Kendall-Taylor
P
1986
Growth hormone secreting pituitary adenomas are heterogeneous in cell culture and commonly secrete glycoprotein hormone α-subunit.
Clin Endocrinol (Oxf)
25
:
173
–
179
40
Oppenheim
DS
,
Kana
AR
,
Sangha
JS
,
Klibanski
A
1990
Prevalence of α-subunit hypersecretion in patients with pituitary tumors: clinically nonfunctioning and somatotroph adenomas.
J Clin Endocrinol Metab
70
:
859
–
864
41
Shimon
I
,
Nass
D
,
Gross
DJ
2001
Pituitary macroadenoma secreting thyrotropin and growth hormone: remission of bihormonal hypersecretion in response to lanreotide therapy.
Pituitary
4
:
265
–
269
42
Mohammad
HP
,
Abbud
RA
,
Parlow
AF
,
Lewin
JS
,
Nilson
JH
2003
Targeted overexpression of luteinizing hormone causes ovary-dependent functional adenomas restricted to cells of the Pit-1 lineage.
Endocrinology
144
:
4626
–
4636
43
Abbud
RA
,
Ameduri
RK
,
Rao
JS
,
Nett
TM
,
Nilson
JH
1999
Chronic hypersecretion of luteinizing hormone in transgenic mice selectively alters responsiveness of the α-subunit gene to gonadotropin-releasing hormone and estrogens.
Mol Endocrinol
13
:
1449
–
1459
44
Santos
AF
,
Huang
H
,
Tindall
DJ
2004
The androgen receptor: a potential target for therapy of prostate cancer.
Steroids
69
:
79
–
85
45
Oliver
SE
,
Barrass
B
,
Gunnell
DJ
,
Donovan
JL
,
Peters
TJ
,
Persad
RA
,
Gillatt
D
,
Neal
DE
,
Hamdy
FC
,
Holly
JM
2004
Serum insulin-like growth factor-I is positively associated with serum prostate-specific antigen in middle-aged men without evidence of prostate cancer.
Cancer Epidemiol Biomarkers Prev
13
:
163
–
165
46
Konno-Takahashi
N
,
Takeuchi
T
,
Shimizu
T
,
Nishimatsu
H
,
Fukuhara
H
,
Kamijo
T
,
Moriyama
N
,
Tejima
S
,
Kitamura
T
2003
Engineered IGF-I expression induces glandular enlargement in the murine prostate.
J Endocrinol
177
:
389
–
398
47
Cohen
P
,
Clemmons
DR
,
Rosenfeld
RG
2000
Does the GH-IGF axis play a role in cancer pathogenesis?
Growth Horm IGF Res
10
:
297
–
305
48
DiGiovanni
J
,
Kiguchi
K
,
Frijhoff
A
,
Wilker
E
,
Bol
DK
,
Beltran
L
,
Moats
S
,
Ramirez
A
,
Jorcano
J
,
Conti
C
2000
Deregulated expression of insulin-like growth factor 1 in prostate epithelium leads to neoplasia in transgenic mice.
Proc Natl Acad Sci USA
97
:
3455
–
3460
49
Ruan
W
,
Powell-Braxton
L
,
Kopchick
JJ
,
Kleinberg
DL
1999
Evidence that insulin-like growth factor I and growth hormone are required for prostate gland development.
Endocrinology
140
:
1984
–
1989
50
Romero
F
,
Gil-Bernabe
AM
,
Saez
C
,
Japon
MA
,
Pintor-Toro
JA
,
Tortolero
M
2004
Securin is a target of the UV response pathway in mammalian cells.
Mol Cell Biol
24
:
2720
–
2733
51
Hamid
T
,
Kakar
SS
2004
PTTG/securin activates expression of p53 and modulates its function.
Mol Cancer
3
:
18
52
Bernal
JA
,
Luna
R
,
Espina
A
,
Lazaro
I
,
Ramos-Morales
F
,
Romero
F
,
Arias
C
,
Silva
A
,
Tortolero
M
,
Pintor-Toro
JA
2002
Human securin interacts with p53 and modulates p53-mediated transcriptional activity and apoptosis.
Nat Genet
32
:
306
–
311
53
Wang
Z
,
Melmed
S
2000
Pituitary tumor transforming gene (PTTG) transforming and transactivation activity.
J Biol Chem
275
:
7459
–
7461
54
Ren
SG
,
Kim
S
,
Taylor
J
,
Dong
J
,
Moreau
JP
,
Culler
MD
,
Melmed
S
2003
Suppression of rat and human growth hormone and prolactin secretion by a novel somatostatin/dopaminergic chimeric ligand.
J Clin Endocrinol Metab
88
:
5414
–
5421
Copyright © 2005 by The Endocrine Society
Citations
Views
Altmetric
Metrics
Total Views 803
568 Pageviews
235 PDF Downloads
Since 3/1/2017
Month: | Total Views: |
---|---|
March 2017 | 1 |
April 2017 | 2 |
May 2017 | 1 |
June 2017 | 1 |
July 2017 | 3 |
August 2017 | 2 |
September 2017 | 5 |
October 2017 | 1 |
November 2017 | 2 |
December 2017 | 5 |
January 2018 | 7 |
February 2018 | 11 |
March 2018 | 16 |
April 2018 | 21 |
May 2018 | 18 |
June 2018 | 22 |
July 2018 | 9 |
August 2018 | 18 |
September 2018 | 10 |
October 2018 | 3 |
November 2018 | 18 |
December 2018 | 15 |
January 2019 | 4 |
February 2019 | 13 |
March 2019 | 6 |
April 2019 | 11 |
May 2019 | 8 |
June 2019 | 16 |
July 2019 | 17 |
August 2019 | 10 |
September 2019 | 15 |
October 2019 | 11 |
November 2019 | 4 |
December 2019 | 11 |
January 2020 | 7 |
February 2020 | 6 |
March 2020 | 11 |
April 2020 | 11 |
May 2020 | 5 |
June 2020 | 12 |
July 2020 | 7 |
August 2020 | 3 |
September 2020 | 9 |
October 2020 | 3 |
November 2020 | 4 |
December 2020 | 8 |
January 2021 | 6 |
February 2021 | 5 |
March 2021 | 12 |
April 2021 | 4 |
May 2021 | 3 |
June 2021 | 1 |
July 2021 | 2 |
August 2021 | 3 |
September 2021 | 9 |
October 2021 | 3 |
November 2021 | 12 |
December 2021 | 5 |
January 2022 | 4 |
February 2022 | 1 |
March 2022 | 5 |
April 2022 | 8 |
May 2022 | 8 |
June 2022 | 6 |
July 2022 | 22 |
August 2022 | 11 |
September 2022 | 12 |
October 2022 | 8 |
November 2022 | 4 |
December 2022 | 5 |
January 2023 | 17 |
February 2023 | 9 |
March 2023 | 9 |
April 2023 | 7 |
May 2023 | 9 |
June 2023 | 15 |
July 2023 | 3 |
August 2023 | 3 |
September 2023 | 13 |
October 2023 | 14 |
November 2023 | 19 |
December 2023 | 14 |
January 2024 | 5 |
February 2024 | 19 |
March 2024 | 9 |
April 2024 | 20 |
May 2024 | 11 |
June 2024 | 11 |
July 2024 | 15 |
August 2024 | 4 |
September 2024 | 5 |
October 2024 | 10 |
Citations
88 Web of Science
×
Email alerts
Related articles in PubMed
Citing articles via
More from Oxford Academic