D-type cyclins (Dl, D2, and D3) have been implicated in the regulation of G] progression, cell cycle withdrawal, and differentiation in numerous cell types. Therefore, in addition to cyclin E expression, we examined the expression of cyclins Dl, D2, and D3 in freshly isolated and cultured trophoblasts and proliferating ED27 and JEG-3 cells, as well as in MCF-7 breast cancer cells. As shown in Figure 2a, cyclin E expression was greatest in freshly isolated trophoblasts and trophoblasts cultured for 24 h (Fig. 3a). asthma inhalers
Cyclin E protein was present in MCF-7 cells but was expressed at levels 5-fold below that in trophoblasts and JEG-3 cells (Fig. 3a). Western blot analysis revealed expression of cyclin Dl protein only in proliferating MCF-7 cells (Fig. 3b). There was no detectable cyclin Dl expression in trophoblasts or cell lines originating from trophoblasts. Identical results were observed in five separate placentae. Cyclin D2 expression was visible in first-trimester ED27 cells and MCF-7 cells and faintly evident in freshly isolated cytotrophoblasts (Fig. 3c). Strong expression of cyclin D3 protein was present in freshly isolated trophoblasts, proliferating ED27 and JEG-3 cells, and MCF-7 cells (Fig. 3d). Cultured trophoblasts at 24 and 48 h postplating demonstrated declining cyclin D3 expression, and by 72 h, cyclin D3 expression was undetectable (Fig. 3d). Levels of Cdk4, the catalytic partner of D-type cyclins, did not change throughout the culture period (data not shown).
FIG. 3. Differential expression of D-type cyclins in term trophoblastand proliferating cell lines. Normal trophoblasts (CT), JEG-3 (J), ED27 (E), and MCF-7 (M) cells were prepared for immunobiotting as described in Materials and Methods. Western blot analysis of cyclins E, D1, D2, and D3 for cytotrophoblasts and cultured trophoblasts (CT; Time = 0, 24, 48, 72, and 96 h).