A model for the mechanism of DNA damage and cell death induced by Plk1 depletion. Under normal conditions, Plk1 phosphorylates Emi1, which results in degradation of Emi1 and activation of APC/C. Activated APC/C degrades the mitotic proteins, such as cyclin B1 and geminin, at the end of mitosis leading to exit from M phase. Upon Plk1 depletion, Emi1 is not phosphorylated by Plk1, which results in the accumulation of Emi1. The high level of Emi1 inhibits the APC/C activity, which leads to a high level of geminin, an inhibitor of prereplicative complex. Consequently, geminin disrupts the pre-RC and DNA damage-sensing kinases are activated.

DNA damage occurs in G₁/early S phase in HeLa cells. HeLa cells were synchronized with double thymidine block and infected with lentivirus expressing RNAi targeting Plk1 (Plk1−) or with control (Con) virus. (A and B) Cells were released with fresh medium in the presence (+) or absence of hydroxyurea (HU). Cells with DNA damage are visualized by the comet assay and quantified. Bars, 50 μm. (C) FACS analysis of the cells shown in panels A and B. (D) The γ-H2A.X-positive cells were counted and quantified at the indicated times.

The involvement of p53 and p73 in DNA damage induced by Plk1 depletion. Cells were synchronized with double thymidine block and infected with lentivirus expressing RNAi targeting Plk1 (Plk1−) or with control (Con) virus. (A and B) The γ-H2A.X- or cleaved caspase-3-positive cells were counted and quantified. (C) FACS analysis of cell cycle progression in Plk1-depleted H1299 cells. (D) Extracts of H1299 cells were subjected to immunoblot analysis with anti-p-Chk2 (T68), anti-E2F1, anti-p73, anti-p-Chk1 (S345), anti-Plk1, and anti-Erk2 antibodies. (E and G) H1299 cells were transfected with human p73 siRNA and infected with lentivirus targeting human Plk1 during the double thymidine block as described in Materials and Methods. (E and F) At 12 h and 24 h after release from the double thymidine block, cells were prepared, and FACS analysis was performed. The subgenomic DNA was quantified. (G) At 12 h after release from the double thymidine block, extracts were subjected to immunoblot analysis with anti-p73, anti-Plk1, anti-Bax, and anti-Erk2 antibodies.

The formation of γ-H2A.X foci precedes activation of caspase-3 in Plk1-depleted HeLa cells. HeLa cells were synchronized with double thymidine block and infected with either lentivirus expressing RNAi targeting Plk1 (Plk1−) or with control virus as described in Materials and Methods. Note that zero time of release is 24 h after infection. (A) Whole-cell lysates were subjected to immunoblot analysis with anti-Plk1 and anti-Erk2 antibodies. (B) FACS analysis of cell cycle progression. Con, control. (C) HeLa cells grown on coverslips were fixed with 4% paraformaldehyde and examined for phosphorylated H2A.X (green) or cleaved caspase-3 (red). Nuclear DNA was stained with DAPI. Bars, 10 μm. 1D and 2D, 1 day and 2 days after infection, respectively. (D and E) The γ-H2A.X- and cleaved caspase-3-positive [caspase-3- (+)] cells were counted and quantified.

Plk1 depletion results in high levels of Emi1 and geminin. (A) Plk1-depleted (Plk1−) and control (Con) HeLa cells were synchronized and infected as described in Materials and Methods. Whole-cell lysates were subjected to immunoblot analysis with anti-Emi1, antigeminin, anti-cyclin B1, anti-Plk1, anti-Erk2, anti-p-ATM (S1981), anti-p-Chk2 (T68), and anti-Chk2 antibodies. (B) HeLa cells grown on coverslips were infected, synchronized, and transfected as described in the legend to Fig. 4D. At 2 h after release, cells were fixed with 4% paraformaldehyde and examined for GFP (green) or Emi1 (red). Nuclear DNA was stained with DAPI. Bars, 10 μm. (C) HeLa cells were synchronized and infected as described in Materials and Methods. At 2 h after release, cells were fractionated into soluble and chromatin fractions and subjected to immunoblotting with anti-Cdt1, antigeminin, anti-Plk1, and anti-Erk2 antibodies. (D) The soluble extracts of cells infected with lentivirus expressing RNAi targeting Plk1 (P) or control virus (C) were subjected to immunoprecipitation (IP) assay using antigeminin antibody and subjected to immunoblotting with anti-Cdt1 and antigeminin antibodies. IgG, immunoglobulin G.

Plk1 depletion inhibits DNA synthesis and disrupts the binding of Mcm proteins to chromatin in early S phase. HeLa cells were synchronized and infected as described in Materials and Methods. Plk1-depleted (Plk1−) and control (Con) cells were used. (A and B) At 1 h or 1 h 30 min after release, BrdU was added to the cells, and after 60 min or 30 min, the cells were fixed and labeled with anti-BrdU antibody (green). Nuclear DNA was stained with DAPI. Bars, 50 μm. The BrdU-positive [(+)] cells were counted and quantified. (C) At 2 h after release, cells were fractionated into soluble and chromatin fractions as described in Materials and Methods and subjected to immunoblotting with anti-Mcm7, anti-Mcm3, antinucleophosmin, anti-Plk1, and anti-Erk2 antibodies. NPM, nucleophosmin. (D) HeLa cells were infected with Plk1-targeting lentivirus or control virus. After 8 h, cells were treated with thymidine for 16 h and then transfected with pEGFP (GFP), pEGFP-Plk1-WT (WT), or pEGFP-Plk1-K82M (KM). Murine Plk1 is resistant to human Plk1 RNAi. After 8 h, the cells were again treated with thymidine for 16 h and then released with fresh medium. At 2 h after release, cells were fractionated into soluble and chromatin fractions and subjected to immunoblotting with anti-Mcm7, antinucleophosmin, anti-Plk1, and anti-Erk2 antibodies. (E) At 2 h after release, extracts were subjected to ATM or ATR assay using anti-ATM antibody or anti-ATR antibody, respectively. The kinase activity was measured with PHAS-1 as a substrate. PHAS-1 was visualized by staining with Coomassie brilliant blue. Ig G, immunoglobulin G.

DNA damage occurred in the G₁/S transition, and DNA synthesis was reduced in Plk1-depleted T98G cells. T98G cells were synchronized by serum starvation for 2 days and infected with lentivirus expressing RNAi targeting Plk1 (Plk1− or P) or with control virus (Control, Con, or C) in serum-free medium for 1 day. Cells were released with fresh medium containing 10% fetal bovine serum and then harvested at various times. (A) Extracts were subjected to ATM assay using anti-ATM antibody with PHAS-1 as the substrate. PHAS-1 was visualized by staining with Coomassie brilliant blue. Also immunoblot (IB) analysis was performed with anti-Plk1 and anti-Erk2 antibodies. IgG, immunoglobulin G. (B) DNA damage was measured by immunohistochemistry using γ-H2A.X monoclonal antibody. The γ-H2A.X-positive cells were counted and quantified. (C) FACS analysis was performed. (D) At 16 h after release, BrdU was added to the cells. After 30 min, cells were fixed and labeled by anti-BrdU antibody (green). Nuclear DNA was stained by DAPI. Bars, 20 μm. (E) The BrdU-positive [BrdU(+)] T98G cells were counted and quantified. (F) T98G cells were synchronized with serum starvation for 2 days and infected as described above. Whole-cell lysates were subjected to immunoblot analysis using anti-p-ATM (S1981), anti-Emi1, antigeminin, anti-Plk1, and anti-Erk2 antibodies. (G) T98G cells were synchronized with double thymidine block and infected with lentivirus as described above. Lysates were subjected to immunoblot analysis using anti-p-Chk2 (T68), anti-Emi1, anti-Plk1, and anti-Erk2 antibodies.