In: Progesterone7 Feb 2013
Stromal cells cultured in serum-free medium for 72 h were stimulated to synchronously reenter the cell cycle by addition of basic fibroblast growth factor (bFGF) and progesterone. It should be kept in mind, as the flow cytometry analyses showed (Fig. 1), that all of the cells do not require stimulation into the cell cycle because perfect synchrony is never achieved using cultured cells. However, progesterone and bFGF significantly (p < 0.01) increased stromal cell proliferation in a dose-dependent fashion. Of the growth factors tested in this culture system, epidermal growth factor, transforming growth factor a, and bFGF stimulated stromal cell division significantly (p < 0.01) over that in the control cultures containing serum-free medium alone. None of the growth factors stimulated proliferation significantly in the absence of progesterone, and addition of two growth factors did not stimulate proliferation as compared to progesterone with growth factor. This progesterone-dependent proliferation was specific, because dexamethasone, estradiol-17p, and 5a-dihydrotestos-terone did not stimulate proliferation significantly. buy levaquin online
More recently, we demonstrated that cells treated with bFGF or progesterone alone are unable to progress through G1 and enter DNA replication (Fig. 2). Stromal cells cultured with progesterone and bFGF incorporated [3H]thymidine significantly (p < 0.01) compared to cells treated with progesterone alone, fibroblast growth factor (FGF) alone, or serum-free medium (control) (Fig. 2). To ensure that stromal cells were progressing through the cell cycle, the number of cells over the stimulation period was counted (Fig. 3). Cell number increased slightly at 24 h in cultures containing serum-free medium and serum-free medium with progesterone and bFGF. However, this increase was significantly greater (p < 0.01) at 48 h in cell cultures containing progesterone and bFGF as compared with the control cultures. Analysis of cell cycle phases using flow cytometry showed that the accumulation of cells in S phase, at the expense of a reduction of cells in G1 phase, occurred 24 h after addition of bFGF and progesterone (Fig. 4). These results are consistent with a single stromal cell transit through the cell cycle resulting in the 2-fold increase in cell number over this same time period (compare Figs. 3 and 4). Taken together, these data indicate that progesterone and bFGF stimulate mitosis and not just the rescue of cell death.
FIG. 2. Synergistic action between progesterone and bFGF stimulates stromal cell progression through G1 and entry into DNA replication. Quiescent uterine stromal cells (UIII, passage 18) were stimulated with either FGF (50 ng/ml), medroxyprogesterone acetate (MPA, 1 ^M), the two agents together (FGF+MPA), or serum-free medium alone (Control) for 24 h. The cells were pulsed for 2 h with [3H]thymidine (1 ^Ci/well). Incorporation of [3H]thymidine was determined by scintillation counting. Results are expressed as the mean ± SEM for 6 independent observations for each treatment. *p < 0.01.
FIG. 3. Progesterone and bFGF stimulate stromal cell proliferation. Serum-starved stromal cells (passages 17-20) were stimulated with 1 ^M progesterone and 50 ng/ml bFGF (dashed line) or were provided medium with vehicle alone (solid line). The number of cells was counted at 0, 24, and 48 h after mitogen addition. The Time 0 was determined from the basal number of cells at the time of progesterone and bFGF addition. After 48 h of culture, cell number increased significantly (p < 0.01) in cell cultures treated with bFGF and progesterone over that in the control conditions. Data shown are the mean ± SEM of triplicate samples.
FIG. 4. Temporal analysis of cell cycle phases in uterine stromal cells. Cells (UII, passage 17) were synchronized by culture in serum-free medium. The cells were stimulated to reenter the cell cycle by addition of serum-free medium containing bFGF (50 ng/ml) and progesterone (1 ^M). Cells were collected at the indicated times, and cell cycle progression was monitored by flow cytometry.
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