Induction of DNA synthesis in dog thyrocytes in primary culture: Synergistic effects of thyrotropin and cyclic AMP with epidermal growth factor and insulin

Abstract

We have investigated the growth effects of thyrotropin (TSH) (mimicked by forskolin and acting through cyclic AMP), epidermal growth factor (EGF), serum (10%) and insulin on quiescent dog thyroid epithelial cells in primary culture in a serum-free defined medium. These cells were previously shown to retain the capacity to express major thyroid differentiation markers. In the presence of insulin and after a similar prereplicative phase of 18 +/- 2h, TSH, EGF, and serum promoted DNA synthesis in such quiescent cells only a minority of which had proliferated in vitro before stimulation. The combination of these factors induced more than 90% of the cells to enter S phase within 48 h and near exponetial proliferation. Analysis of the cell cycle parameters of the stimulated cells revealed that the G1 period duration was similar to the length of the prereplicative phase of quiescent thyroid cells; this might indicate that they were in fact in an early G1 stage rather than in G0 prior to stimulation. TSH and EGF action depended on or was potentiated by insulin. Strikingly, nanomolar concentrations of insulin were sufficient to support stimulation of DNA synthesis by TSH, while micromolar concentrations of insulin were required for the action of EGF. This suggests that insulin supported the action of TSH by acting on its own high affinity receptors, whereas its effect on EGF action would be related to its somatomedinlike effects at high supraphysiological concentrations. Insulin stimulated the progression in the prereplicative phase initiated by TSH or forskolin. In addition, in some primary cultures TSH must act together with insulin to stimulate early events of the prereplicative phase. In the presence of insulin, EGF, and forskolin, an adenylate cyclase activator, markedly synergized to induce DNA synthesis. Addition of forskolin 24 h after EGF or EGF 24 h after forskolin also resulted in amplification of the growth response but with a lag equal to the prereplicative period observed with the single compound. This indicates that events induced by the second factor can no longer be integrated during the prereplicative phase set by the first factor. These findings demonstrate the importance of synergistic cooperation between hormones and growth factors for the induction of DNA synthesis in epithelial thyroid cells and support the proposal that essentially different mitogenic pathways--cyclic AMP-dependent or independent--may coexist in one cell.

Nature of the Critical Labile Event That Controls RB Phosphorylation in the Cyclic AMP-Dependent Cell Cycle of Thyrocytes in Primary Culture

“…In response to TSH or forskolin quiescent dog thyrocytes cultured in the presence of insulin progressively enter DNA synthesis phase after about 18 h [24,25] (Fig. 1).…”

“…Follicular cells were obtained from dog thyroid tissue as described [24]; cultured in monolayer in the "control" medium, DMEM ϩ Ham's F12 ϩ MCDB104 medium (Gibco Laboratories, Paisley, UK; 2:1:1 by vol.) supplemented with bovine insulin (Sigma; 5 g/ml), ascorbic acid (40 g/ml), and antibiotics [24]; and stimulated at day 4 with forskolin (Calbiochem; 2 ϫ 10 Ϫ6 M) as detailed in the figure legends.…”

“…supplemented with bovine insulin (Sigma; 5 g/ml), ascorbic acid (40 g/ml), and antibiotics [24]; and stimulated at day 4 with forskolin (Calbiochem; 2 ϫ 10 Ϫ6 M) as detailed in the figure legends. Immunofluorescent detections of proliferating cell nuclear antigen (PCNA) [25], cyclin D3, and CDK4 [22] were performed exactly as described from cells fixed with 2% paraformaldehyde for 90 s and then with methanol for 10 min at Ϫ20°C.…”

Effect of Monensin on DNA Synthesis in Cultured Human Thyroid Cells.

“…However, the growth promoting effect of TSAb or TGI remains controversial [6][7][8][9][10]. EGF or other factors such as insulin, IGF-1, or interleukin-1 also stimulate the growth of thyroid follicular cells [11][12][13][14][15][16][17][18][19][20]. Several investigators claimed that TGI was actually a contamination of EGF [8,9].…”

“…Several investigators claimed that TGI was actually a contamination of EGF [8,9]. Moreover, the signal transduction pathway of these growth factors remains unclear [3][4][5][11][12][13][14][15][16][17][18][19][20].…”