p21(Cip1) restrains pituitary tumor growth

Abstract

As commonly encountered, pituitary adenomas are invariably benign. We therefore studied protective pituitary proliferative mechanisms. Pituitary tumor transforming gene (Pttg) deletion results in pituitary p21 induction and abrogates tumor development in Rb(+/-)Pttg(-/-) mice. p21 disruption restores attenuated Rb(+/-)Pttg(-/-) pituitary proliferation rates and enables high penetrance of pituitary, but not thyroid, tumor growth in triple mutant animals (88% of Rb(+/-) and 72% of Rb(+/-)Pttg(-/-)p21(-/-) vs. 30% of Rb(+/-)Pttg(-/-) mice developed pituitary tumors, P < 0.001). p21 deletion also accelerated S-phase entry and enhanced transformation rates in triple mutant MEFs. Intranuclear p21 accumulates in Pttg-null aneuploid GH-secreting cells, and GH(3) rat pituitary tumor cells overexpressing PTTG also exhibited increased levels of mRNA for both p21 (18-fold, P < 0.01) and ATM (9-fold, P < 0.01). PTTG is abundantly expressed in human pituitary tumors, and in 23 of 26 GH-producing pituitary adenomas with high PTTG levels, senescence was evidenced by increased p21 and SA-beta-galactosidase. Thus, either deletion or overexpression of Pttg promotes pituitary cell aneuploidy and p53/p21-dependent senescence, particularly in GH-secreting cells. Aneuploid pituitary cell p21 may constrain pituitary tumor growth, thus accounting for the very low incidence of pituitary carcinomas.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

p21 deletion from the Rb+/−Pttg−/− background restores pituitary but not thyroid tumor development in mice. (A) Kaplan–Meier survival analysis (log-rank test) of the time of death with evidence of pituitary tumor showed significant differences between Rb+/−Pttg−/− and Rb+/−Pttg+/+ (P < 0.001), between Rb+/−Pttg−/− and Rb+/+Pttg−/− (P < 0.05), and between a Rb+/−Pttg−/− and Rb+/−Pttg−/−p21−/− mice (P = 0.007). (B) Kaplan–Meier survival analysis (log-rank test) with evidence of thyroid tumors in the different genotypes showed significant differences between Rb+/−Pttg−/− and Rb+/−Pttg+/+ (P < 0.001), and between Rb+/−Pttg+/+ and Rb+/−Pttg−/−p21−/− mice (P < 0.001). No differences were observed between Rb+/−Pttg−/− and Rb+/−Pttg−/−p21−/− mice. n, number of animals killed. Western blot analysis of proliferation markers in (C) pituitary and (D) spleen and lung lysates derived from Rb+/− Pttg−/− and Rb+/− Pttg−/− p21−/− mice.

Fig. 2.

Pituitary p21 expression. Fluorescence immunohistochemistry of p21 expression in WT (A) and Pttg−/− (B) pituitary glands. High resolution (×100) confocal image shows intranuclear p21 expression in Pttg−/− pituitary anterior (C) and intermediate (D) lobes. Paraffin slides were labeled with p21 antibody (green). Here and below slides were counterstained with DNA-specific dye ToPro3 (blue). (E) Pttg deletion evokes aneuploidy and enhances p21 expression in GH-producing pituitary cells. Confocal image of Pttg-null pituitary tissue labeled with p21 antibody (green) and GH- antibody (red). (F) The same as E but stained with ACTH antibody (red). In E and F, arrows indicate aneuploid nuclei expressing p21. (G) Percent of hormone-producing cells coexpressing intra-nuclear p21 in Pttg−/− glands.

Fig. 3.

(A–C) Pttg−/− MEFs exhibit abnormal nuclear morphology, DNA damage, and activation of the p53/p21 senescence pathway. (A) Hematoxylin and eosin staining of 1st passage WT and Pttg−/− MEFs. (B) Western blot analysis of p53/p21 senescence pathway markers in 10th passage MEFs. (C) Western blot analysis of DNA damage/repair proteins. Experiments were repeated 3 times with similar results, and representative blots are shown. (D–F) p21 deletion results in increased cell proliferation, accelerated G1-S phase transition, and enhanced transformation. (D)12th passage MEFs (3 × 105) were plated in duplicate 10-cm dishes and cells counted daily for 5 days. (E) 8th passage MEFs were synchronized in 0.1% FBS for 72 h and then cultured in 10% FBS. At the indicated times, duplicate samples were pulsed with BrdU for 30 min and analyzed by flow cytometry and S-phase cells were identified by staining with BrdU antibodies. (F) Proliferation of 12th passage MEFs from 5 experimental genotypes. In D–F, the cell number at each time-point represents the average of duplicate plates ± range. These experiments were performed with 2 independent MEF preparations with similar results, and a single experiment is depicted. (G) 4th passage MEFs derived from the indicated genotypes were infected with retrovirus encoding Ras+T-Ag and cultured in 6-well plates in triplicate for 21 days, and the number of colonies per well was counted. Numbers of colonies are expressed as mean ± SE of 3 independent MEF preparations. (H) γ-foci in 10th passage MEF nuclei. Depicted are single representative high-resolution confocal images of asynchronous and untreated mouse MEFs fixed and labeled with anti γ-H2AX antibody. Foci were counted and measured in 30 to 40 nuclei of each genotype, and focus number and size were determined by using ImageJ Software (http://rsb.info.nih.gov/ij).

Fig. 4.

Both Pttg overexpression and silencing induces p21 in GH3 cells. (A) Confocal image of double fluorescence immunohistochemistry of rat GH-producing GH3 cells transfected with EGFP or EGFP-PTTG. Cells were fixed, labeled with EGFP antibody (green), p21 antibody (red). Cells coexpressing EGFP-PTTG and p21 appeared white. (B and C) Pttg overexpression induced p21 mRNA and ATM mRNA levels in GH3 cells. Cells were transfected with plasmids expressing EGFP or EGFP-PTTG and sorted by flow cytometry, and real-time PCR of p21 mRNA and ATM mRNA was performed. (D) Pttg silencing by Pttg siRNA induces p21 mRNA levels detected by real-time PCR. Values are expressed as mean ± SE of triplicate measurements for each experimental group. All experiments were repeated twice, and representative experiments are shown. *, P < 0.05 vs. control.

Fig. 5.

p21 is induced in human GH-producing pituitary adenomas. (A) (a–e) Immunohistochemistry of pituitary adenoma sections stained for p21 (brown). (f) Normal pituitary. (g) Breast carcinoma. (h) Pituitary carcinoma. (B) Confocal image of fluorescence immunohistochemistry for p21 and PTTG expression. Specimen was labeled with p21 antibody (red) and PTTG antibody (green). (C and D) Reciprocal p21 and PTTG expression in all pituitary tumor types (C) and in GH-secreting adenomas (D).

Fig. 6.

Senescence markers in human GH-producing pituitary adenomas. (A) Immunohistochemistry of the same GH-secreting human adenoma sections stained for p21 (brown) and SA-β-gal activity (blue). (B) Confocal image of double fluorescence immunohistochemistry of p21 (green) and β-galactosidase (red) proteins coexpression in human pituitary adenomatous but not in normal adjacent tissue (Left). High resolution (×63) image of the same slide (Right). (C) Proposed model for p21-induced senescence in the hypoplastic Pttg-null pituitary gland and PTTG-overexpressing pituitary adenomas. Arrows depict proposed pathways.