Expression of D-type cyclins in normal and neoplastic rat pituitary.

Abstract

The D-type cyclins (D1, D2, and D3) are involved in progression through the G1 phase of the cell cycle and are induced as part of the delayed early response to growth factor stimulation. To better understand the role of D-type cyclins in pituitary cell function and the regulatory role of growth factors in the cell cycle, we analyzed the expression and regulation of D-type cyclins in normal and neoplastic rat pituitary cells. Immunocytochemical and RT-PCR analyses showed expression of all three D-type cyclins in the normal pituitary, with higher percentages of positive cells by immunocytochemistry in the nuclei of normal pituitaries (D1, 20-30%; D2, 50-60%; D3, 70-80%), compared with GH3 cells. In the normal pituitary, there were significantly higher levels of cyclins D2 and D3 in PRL, GH, LH, and TSH cells, compared with ACTH cells. Cyclin D1 protein was not detected in GH3 cells, while D2 was present in less than 1 percent and D3 in 10-15 percent of GH3 cells. There were low levels of cyclin D1 and D2 messenger RNA expression in GH3 cells, by RT-PCR. When dissociated rat pituitary cells were cultured in the presence of basic fibroblast growth factor (5.6 nM) for 3 days, cyclin D2 was up-regulated 2-fold in normal PRL cells (control, 33 +/- 1%; treated, 68 +/- 2%). Similarly, bFGF treatment stimulated cyclin D3 expression 3-fold in GH3 cells (control, 15 +/- 1%; treated, 44 +/- 1%). Treatment of GH3 cells with 5-aza-2'-deoxycytidine, which induces gene demethylation, produced marked increases in cyclin D2 and D3 expression. Transfection of mouse cyclin D1 complementary DNA, driven by a cytomegalovirus promoter into GH3 cells, led to ectopic cyclin D1 expression; and there was a slight stimulation of cell proliferation and increased apoptosis in GH3 cells. These results indicate that there is a differential expression of various D-type cyclins in different types of normal pituitary cells and between normal pituitary and GH3 cells. Growth factors, such as bFGF and demethylation increased D-type cyclin expression, whereas ectopic overexpression of cyclin D1 stimulates cell proliferation and increases apoptosis in GH3 pituitary tumor cells.