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. 2003 May 13;100(10):5789-94.
doi: 10.1073/pnas.1031523100. Epub 2003 May 5.

Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells

Xiaoqi Liu  1 , Raymond L Erikson
Affiliations

Polo-like kinase (Plk)1 depletion induces apoptosis in cancer cells

Xiaoqi Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Elevated expression of mammalian polo-like kinase (Plk)1 occurs in many different types of cancers, and Plk1 has been proposed as a novel diagnostic marker for several tumors. We used the recently developed vector-based small interfering RNA technique to specifically deplete Plk1 in cancer cells. We found that Plk1 depletion dramatically inhibited cell proliferation, decreased viability, and resulted in cell-cycle arrest with 4 N DNA content. The formation of dumbbell-like chromatin structure suggests the inability of these cells to completely separate the sister chromatids at the onset of anaphase. Plk1 depletion induced apoptosis, as indicated by the appearance of subgenomic DNA in fluorescence-activated cell-sorter (FACS) profiles, the activation of caspase 3, and the formation of fragmented nuclei. Plk1-depletion-induced apoptosis was partially reversed by cotransfection of nondegradable mouse Plk1 constructs. In addition, the p53 pathway was shown to be involved in Plk1-depletion-induced apoptosis. DNA damage occurred in Plk1-depleted cells and inhibition of ATM strongly potentiated the lethality of Plk1 depletion. Although p53 is stabilized in Plk1-depleted cells, DNA damage also occurs in p53(-/-) cells. These data support the notion that disruption of Plk1 function could be an important application in cancer therapy.

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Figures

Figure 1
Figure 1
Vector-based siRNA targeting Plk1. (A) HeLa cells were transfected with pBS/U6-Plk1 only (without cotransfection of pBabe-puro). At 2 d posttransfection, cells were harvested, and cell lysates were subjected to direct Western blotting using the antibodies as indicated on the left. (B and C) Plk1 is required for cell growth and survival. HeLa cells were transfected as described in Materials and Methods, and cell proliferation (B) and viability (C) were monitored. To determine cell viability, floating cells and attached cells were harvested and counted separately. Viability was calculated as the percentage of attached cells compared to total cells.
Figure 2
Figure 2
Plk1 depletion induces G2/M arrest and apoptosis. After transfection as described in the text, HeLa cells were harvested and analyzed at 3, 4, or 5 d posttransfection as indicated. (A) FACS profiles. The positions of G1, G2/M, and sub-G1 populations are labeled. (B) Western blots using antibodies indicated on the left. (C) HeLa cells on coverslips were transfected as described. At 3 d posttransfection, the cells were fixed and DNA was stained with 4′,6-diamidino-2-phenylindole. Three typical images were normal (Left), dumbbell-like structure (Center), and fragmented nuclei (Right). (Scale bar: 10 μm.) (D) Histogram shows results from five independent experiments (>300 cells each) and bars indicate SD.
Figure 3
Figure 3
Rescue of Plk1-depletion-induced apoptosis by cotransfection of mouse Plk1 constructs. (A) Alignment of Plk1 depletion targeting sequences. The unconserved third positions of four codons between human and mouse Plk1 sequence in the region are indicated as bold. (B) HeLa cells were cotransfected with pBS/U6-Plk1, one of indicated mouse Plk1 constructs and pBabe-puro at the ratio of 6:3:1 by using the protocol described in Materials and Methods. Cells were harvested 5 d posttransfection and cell lysates were analyzed by direct Western blotting using antibodies indicated on the left.
Figure 4
Figure 4
p53 pathway is involved in Plk1-depletion-induced apoptosis events. (A) HeLa cells were transfected as described. At 4 d posttransfection, cells were harvested and cell lysates were subjected to direct Western blotting by using antibodies indicated on the left. (B) Plk1 in HeLa cells was depleted as described above. At indicated times posttransfection, cell lysates were analyzed by anti-caspase 8 Western blotting. Asterisks on the left indicate the positions of cleaved products of caspase 8. Arrowhead on the right indicates the full-length caspase 8 that was mainly cleaved after Plk1 depletion. (C) Immunofluoresence staining of Plk1-depleted cells with phospho-H2AX antibody. (Scale bar: 10 μm.)
Figure 5
Figure 5
ATM inhibition potentiates the lethality of Plk1 depletion. (A and B) HeLa cells were transfected with pBS/U6-Plk1 by using GenePORTER. After overnight incubation, cells were treated with either 2 mM caffeine or 5 μM wortmannin for 1 or 2 additional days. Harvested cells were subjected to FACS analysis (A), and percentage of apoptotic cells was calculated based on the ratio between the number of floating cells versus attached cells (B). (C) ATM−/− (GM05849) cells are hypersensitive to Plk1 depletion. GM05849 cells were transfected with pBS/U6-Plk1 and harvested for FACS analysis at the indicated posttransfection times. The time periods indicated in the figure are the total incubation time posttransfection.
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