Apoptosis in nontumorous and neoplastic human pituitaries: expression of the Bcl-2 family of proteins

Abstract

Analyses of apoptosis and of the apoptosis regulatory proteins Bcl-2, Bax, Bcl-X, and Bad were done in 95 nontumorous and neoplastic pituitary tissues by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL), immunohistochemistry, and Western blotting. The apoptotic index was relatively low in all groups but was at least fourfold higher in pituitary carcinomas compared with any other groups. Pituitaries from pregnant and postpartum women had a fivefold higher apoptotic index compared with matched controls from nonpregnant females. Preoperative treatment of adenomas with octreotide or dopamine agonists did not change the apoptotic index significantly. The lowest levels of Bcl-2, Bax, and Bcl-X expression were in pituitary carcinomas as detected by immunostaining. An immortalized human pituitary adenoma cell line, HP75, developed in our laboratory using a replication-defective recombinant human adenovirus with an early large T-antigen, had a much higher level of apoptosis than nontumorous and neoplastic pituitaries. Treatment with transforming growth factor (TGF)-beta1 and protein kinase C (PKC) inhibitors increased apoptosis in this cell line. Analysis of the Bcl-2 family of proteins after treatment with TGF-beta1 and PKC inhibitors showed a 20% to 30% decrease in Bcl-X in the treated groups compared with controls. These results, which represent the first study of apoptosis in pituitaries from pregnant and postpartum cases and in pituitary carcinomas, indicate that 1) the apoptotic rate is low in nontumorous and neoplastic pituitary tissues but is relatively higher in pituitary carcinomas, 2) there are alterations in the expression of the Bcl-2 family of proteins in pituitary neoplasms with a decrease in Bcl-2 expression in pituitary carcinomas that may contribute to pituitary tumor pathogenesis and/or proliferation, and 3) cultured pituitary tumor cells respond to TGF-beta1 and PKC inhibitors by undergoing apoptotic cell death.

Figure 1.

ACTH adenoma undergoing apoptosis with prominent condensed chromatin and apoptotic body formation in portion of the nucleus. Magnification, ×7500.

Figure 2.

A: Pituitary carcinoma showing two cells with apoptosis in the field shown by dark blue nuclear staining with TUNEL staining. B: The control section in which the terminal deoxynucleotide transferase was omitted was negative for apoptotic cells. Magnification, ×250.

Figure 3.

Analysis of the apoptotic index after TUNEL staining in normal and neoplastic pituitaries. Pituitary carcinomas had a significantly higher apoptotic index than all other groups of tumors and non-neoplastic pituitaries. Apoptotic cells were not detected in prolactinomas treated with dopamine agonist. **P < 0.01 compared with adenomas.

Figure 4.

Immunostaining for Bcl-2 family of proteins in pituitary. A: Normal pituitary showing moderate (2+) diffuse staining for Bax in most anterior pituitary cells. B: GH adenoma with strong staining for Bax in all of the tumor cells. C: ACTH pituitary carcinoma with weak (1+) to moderate (2+) staining for Bax. D: Pituitary for pregnant woman stained for Bcl-X. The folliculate stellate cells that have slender cytoplasmic processes stain strongly for Bcl-X, whereas many of the secretory anterior pituitary cells are staining weakly (1+). The inset shows co-localization of Bcl-x with brown cytoplasmic staining with some S100-protein-positive folliculo-stellate cells with blue nuclear staining. Magnification, ×300 (A), ×250 (B–D), and ×350 (inset).

Figure 5.

Ultrastructural examination of cultured HP75 cells. A: An apoptotic cell with nuclear fragmentation and compact cytoplasm is present. B: An apoptotic body is present in the cell on the left. Magnification, ×5000 (A) and ×6000 (B).

Figure 6.

Effects of TGF-β1 treatment on the apoptotic index (AI) in cultured HP75 cells. The AI increased with duration of treatment between 1 and 3 days using 10⁻⁹ mol/L TGF-β1. Data are from three experiments. **P < 0.01; ***P < 0.001.

Figure 7.

Analysis of the effects of hypericin and chelerythrine chloride treatment on the AI in HP75 cells. Both drugs increased the AI in cultured HP75 cells. Data are from three experiments. *P < 0.05; ***P < 0.001.

Figure 8.

Immunoblot analysis of various Bcl-2 protein family members on HP75 cells after TGF-β1, hypericin, and chelerythrine chloride treatment. Cells were cultured in the presence of the chemicals for 24 hours, and the proteins were extracted and analyzed for specific proteins as indicated. A representative result from two experiments is shown. A: Western blot analysis after enhanced chemiluminescence. Lane 1, control; lane 2, TGF-β1; lane 3, hypericin; lane 4, chelerythrine choloride; lane 5, spleen control tissue. B: Densitometric analysis showing changes in Bcl-2 family of proteins relative to controls.

Figure 9.

Diagramatic summary of proliferative index, apoptotic index, and Bcl-2 expression in normal, hyperplastic, and neoplastic human pituitaries. Both proliferative and apoptotic indices are relatively low in normal, hyperplastic, and adenomatous pituitaries whereas Bcl-2 protein expression is high in these pituitaries. (The data for the proliferative indices for normal pituitary, pituitary adenoma, and carcinomas are from Ref. 14 .) The proliferative index in pituitaries from pregnant women (n = 5) was analyzed in the present study by MIB-1 immunostaining. Pituitary carcinomas, defined by metastatic disease, have much higher proliferative and apoptotic indices and decreased Bcl-2 protein expression indicating that with tumor progression there is increased proliferation as well as increased apoptosis.