Activation of Cdc2/cyclin B and inhibition of centrosome amplification in cells depleted of Plk1 by siRNA

Abstract

The events of the cell cycle, the stages at which the cell proliferates and divides, are facilitated and controlled by multiple signaling pathways. Among the many regulatory enzymes that contribute to these processes is the polo-like kinase (Plk). Plks have been reported to mediate multiple mitotic processes, including bipolar spindle formation, activation of Cdc25C, actin ring formation, centrosome maturation, and activation of the anaphase-promoting complex. To investigate its functions in mammalian cells further, we used the recently developed small interfering RNA technique specifically to deplete Plk1 in cultured cells. We find that Plk1 depletion results in elevated Cdc2 protein kinase activity and thus attenuates cell-cycle progression. About 45% of cells treated with Plk1 small interfering RNA show the formation of a dumbbell-like DNA organization, suggesting that sister chromatids are not completely separated. About 15% of these cells do complete anaphase but do not complete cytokinesis. Finally, Plk1 depletion significantly reduces centrosome amplification in hydroxyurea-treated U2OS cells. These data provide direct evidence that Plk is required for multiple mitotic processes in mammalian cells and their significance is discussed.

Figure 1

Plk1 depletion prevents cyclin B degradation and causes elevated Cdc2 activity. (A) HeLa cells were transfected with RNA oligos against luciferase or Plk1. Thirty-six hours after transfection, cells were treated with 100 ng/ml nocodazole for 12 h and harvested. About 150 μg of cell lysates were directly resolved by SDS/PAGE, and subjected to Western blot by using the antibodies as indicated on the left. (B) Approximately 1 mg of cell lysates were immunoprecipitated with anti-Cdc2 antibody, and the immunoprecipitates were assessed for histone H1 kinase activity. The reaction products were resolved by SDS/PAGE, transferred to membrane, and exposed to the film. The same filter was also probed with anti-Cdc2 antibody to show that equal amounts of proteins were immunoprecipitated.

Figure 2

Plk1 depletion inhibits the separation of sister chromatids. (A) HeLa cells were transfected with RNA oligos against luciferase or Plk1. Forty-eight hours after transfection, cells were fixed and stained with anti-α-tubulin primary antibody, followed with FITC-conjugated anti-mouse secondary antibody. DNA was stained with propidium iodide. (Scale bar: 10 μm.) (B) Histogram shows results from five independent experiments (more than 300 cells each) and bars indicate standard deviations.

Figure 3

Failure of cytokinesis in Plk1-depleted cells. (A) HeLa cells were transfected with a RNA oligo against Plk1. Forty-eight hours after transfection, cells were fixed and stained with anti-α-tubulin primary antibody, followed with FITC-conjugated anti-mouse secondary antibody. DNA was stained with propidium iodide. Three representative images from different phases of cytokinesis are shown. (Scale bar: 10 μm.) (B) Histogram shows results from three independent experiments (more than 300 cells each) and bars indicate standard deviations. An RNA oligo against firefly luciferase gene was used as a control.

Figure 4

Plk1 is involved in centrosome duplication. (A) fluorescence-activated cell sorting profile of U2OS cells with or without hydroxyurea treatment. Positions of cells with 2n or 4n DNA contents are labeled with arrowheads on the bottom. (B) U2OS cells were transfected with RNA oligos against luciferase or Plk1. Twenty-four hours after transfection, cells were treated with 4 mM hydroxyurea (HU) for an additional 40 h (lanes 4–6). For comparison, cells were either untreated (lane 1), treated with mimosine (lane 2), or treated with nocodazole (lane 3). About 300 μg of total cellular protein was used for the direct Western blotting by using the antibodies indicated on the right. Anti-Plk1 Western blot indicates that Plk1 was easily detected in S-phase arrested cells, and treatment with Plk1 siRNA efficiently reduced the Plk1 level. Anti-erk2 Western blot was served as a loading control. (C) Hydroxyurea-treated cells were fixed and stained with anti-γ-tubulin primary antibody, followed with Cy3-conjugated anti-mouse secondary antibody. DNA was stained with 4′,6-diamidino-2-phenylindole. (Scale bar: 10 μm.) (D) Histogram shows results from five independent experiments (more than 300 cells each) and bars indicate standard deviations.