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Silencing of polo-like kinase (Plk) 1 via siRNA causes induction of apoptosis and impairment of mitosis machinery in human prostate cancer cells: implications for the treatment of prostate cancer

Shannon Reagan-Shaw^* and Nihal Ahmad^*,,,1

^* Department of Dermatology,
University of Wisconsin Comprehensive Cancer Center; and
Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin, USA

¹Correspondence: Department of Dermatology, University of Wisconsin, Medical Science Center, 1300 University Avenue, MSC 25B, Madison, WI, 53706, USA. E-mail: nahmad{at}wisc.edu

SPECIFIC AIM

Prostate cancer (PCa) is a major public health concern and a leading cause of cancer-related deaths among males in the U.S. Current therapeutic approaches and surgery options appear to be inadequate for management of PCa. A clear understanding of genetic controls of cellular proliferation and cell division may provide a basis for the rational design of specific targets and therapeutic strategies to manage PCa. Polo-like kinase (Plk) 1 belongs to a family of serine/threonine kinases and has been shown to perform multiple essential functions in cell division and cell cycle progression. The activity of Plk1 has been found to be elevated in tissues and cells with a high mitotic index, including cancer cells, and Plk1 expression levels are believed to have prognostic value for predicting outcomes in patients with some cancers. The role of Plk1 in PCa is not well understood. We propagated a hypothesis that Plk1 plays a critical role in the development of prostate cancer and that the silencing of Plk1 will result in elimination of human PCa cells via inactivation of cyclin-dependent kinase 1 (Cdc2)/cyclin B1-mediated mitotic arrest, followed by apoptosis. Validation of this hypothesis was the major aim of the present study.

PRINCIPAL FINDINGS

1. Small interfering RNA (siRNA) -mediated silencing of Plk1 results in significant inhibition of 1) cell growth and viability, 2) induction of apoptosis, and 3) mitotic arrest in human PCa cells without affecting normal prostate cells
Using the siRNA technique, we determined the effect of a targeted depletion of the Plk1 gene on the viability and growth of three human PCa cell lines (LNCaP, DU145, and PC-3) and normal prostate epithelial (PrEC) cells representing different stages of disease progression. Our data demonstrated that Plk1 protein was abundantly present in PCa cells but virtually undetectable in normal PrEC cells. siRNA-mediated targeted depletion of Plk1 resulted in a significant decrease in 1) endogenous levels of Plk1 protein in PCa cells; 2) viability (45–72% decrease); and 3) growth (19–21%) of PCa cells (data not shown). On the other hand, a similar concentration (100 nM) of Plk1 siRNA had no effect on the viability or growth of normal PrEC cells (data not shown). These observations suggested that Plk1 depletion could be useful in controlling the growth of PCa cells without affecting the normal prostate cells. Our next aim was to determine whether the decrease observed in the viability and growth of PCa cells was mediated by an apoptotic elimination of the cells. As shown in Fig. 1 A–C, transfection of PCa cells with Plk1 siRNA resulted in a significant induction of apoptosis (46–57%) as evident from annexin V binding (Fig. 1A ) and BrdU labeling (Fig. 1B, C ). Transfection of PCa cells with nonsense siRNA at similar concentrations did not affect apoptosis (data not shown). The effect of Plk1 depletion on cell cycle distribution was also determined using a BrdU/PI labeling assay, where Plk1 depletion resulted in a significant accumulation of PCa cells in G2/M phase of the cell cycle (Fig. 1D, E ).

View larger version (41K): [in this window] [in a new window]   Figure 1. Plk1 depletion results in a significant induction of apoptosis and a G2/M phase arrest in PCa cells. A) Determination of apoptosis by confocal microscopy. The cells (DU145, PC-3, and LNCaP) were transfected with Plk1 siRNA, followed by staining with FITC annexin V and PI using Vybrant Apoptosis Assay kit. Annexin V binds to phosphatidylserine, which appears in the outer leaflet of the plasma membrane as a late sign of apoptosis. Apoptotic cells show green fluorescence, necrotic cells show red and green fluorescence, dead cells show red fluorescence, and live cells show no fluorescence. Results are from a representative experiment repeated 3 times with similar results. B) Determination of apoptosis by flow cytometry. After transfection of DU145, PC-3, and LNCaP with Plk1 siRNA, the extent of apoptosis was assessed with the APO-BrdU TUNEL Assay kit. Fragmentation of DNA in apoptotic cells is measured by BrdU incorporation, which is visualized by conjugation to an Alexa Fluor 488 dye-labeled anti-BrdU antibody. Results are from a representative experiment repeated 3 times with similar results. C) Quantitation of apoptosis. The extent of apoptosis was quantified by a computational analysis of cells staining positive for BrdU, using Cell Quest software. The data are expressed as mean ± SE of 3 experiments (*P<0.01). D) Determination of cell cycle distribution. After transfection of DU145, PC-3, and LNCaP with Plk1 siRNA, the cell cycle distribution was assessed with the APO-BrdU TUNEL Assay kit. Results are from a representative experiment repeated 3 times with similar results. E) Quantification of cell cycle distribution. The quantitation of cell cycle distribution was performed using ModFit LT software. Data are expressed as mean ± SE of 3 experiments (*P<0.01).

2. siRNA-mediated silencing of Plk1 results in defects in distribution and integrity of centrosomes and in failure of cytokinesis in human PCa cells
Studies have demonstrated that microinjection of Plk1-specific antibodies resulted in abnormal distribution of condensed chromatin and monoastral microtubule arrays nucleated from duplicated but unseparated chromosomes. To assess the effect of Plk1 depletion on centrosome abnormalities, we immunolabeled the cells with antibodies directed against -tubulin. As shown in Fig. 2 A, B, Plk1 depletion resulted in an abnormal distribution of centrosomes (54–60% defective) in PCa cells. Thus, a high percentage of Plk1 depleted cells showed unseparated chromosomes and multiple centrosomes in a single nuclear membrane, indicating abnormal centrosome distribution in interphase cells. Upon close observation of Plk1-depleted cells by confocal microscopy (Fig. 2C, D ), we found three different populations of chromatin structures in PCa cells: 1) normal chromatin, 2) dumbbell-like (17–20% cells), and 3) fragmented (45–51% cells), indicating defects in centrosome integrity. The appearance of a dumbbell-like DNA structure in Plk1-depleted PCa cells indicated that sister chromatids had not completely separated. Staining of cells with -tubulin confirmed that these connected chromosomes were within one cell, probably in the interphase.

View larger version (22K): [in this window] [in a new window]   Figure 2. Plk1 depletion causes defects in centrosomal distribution, centrosomal abnormalities, and failure of cytokinesis in PCa cells. A) Determination of centrosome distribution. After transfection with Plk1 siRNA in a 2-well culture slide, DU145, PC-3, and LNCaP were labeled with -tubulin antibody and visualized with a Bio-Rad MRC1000 scan head mounted transversely to an inverted Nikon Diaphot 200. An abnormal distribution of centrosomes in cells treated with Plk1 siRNA due to localization of unseparated chromosomes and multiple centrosomes in the same nuclear membrane is shown. Representative pictures from LNCaP cells are shown. B) Quantification of cells with defective distribution of centrosomes. Normal and defective cells in multiple fields were counted under microscope and the data (as % cells with abnormal centrosomes) are shown as a histogram. C) Determination of centrosomal abnormalities. After transfection with Plk1 siRNA in a 2-well culture slide, DU145, PC-3, and LNCaP were labeled with PI and visualized with a Bio-Rad MRC1000 scan head mounted transversely to an inverted Nikon Diaphot 200. Three typical images were normal (C, left), dumbbell-like structure (C, center), or fragmented nuclei (C, right). D) Quantification of cells with centrosomal abnormalities. Normal and defective cells in multiple fields were counted under microscope and the % cells with abnormal centrosomes is shown as a histogram. E) Determination of failure of cytokinesis. After transfection of with Plk1 siRNA in a 2-well culture slide, DU145, PC-3, and LNCaP were labeled with -tubulin and PI and visualized with a Bio-Rad MRC1000 scan head mounted transversely to an inverted Nikon Diaphot 200. The failure of cytokinesis was clearly seen in cells where they were connected by a cytoplasmic bridge as revealed by -tubulin immunofluorescence. F) Quantification of cells with cytokinesis failure. Normal and defective cells in multiple fields were counted under microscope and % cells with cytokinesis failure is shown as a histogram.

We assessed the effect of Plk1 depletion on cytokinesis in PCa cells. As shown in Fig. 2E, F , the targeted depletion of Plk1 resulted in a failure of cytokinesis in 20–25% of PCa cells vs. a very small percentage in untreated cells.

3. siRNA-mediated silencing of Plk1 results in significant increases in Cdc25C, cdc2 and cyclin B1 in PCa cells
Because Plk1 has been shown to function via the Cdc25C and cdc2/cyclin B1 positive feedback loop at the onset of mitosis, we determined the role of Cdc25C and cdc2/cyclin B1 in Plk1 depletion-mediated mitotic arrest of PCa cells. The targeted depletion of Plk1 resulted in a significant increase in Cdc25C, cdc2, phospho-cdc2, and cyclin B1 proteins (data not shown). Studies have shown that phosphorylation of the protein phosphatase Cdc25C by Plk1 promotes nuclear localization of Cdc25C during prophase. In Plk1 depleted PCa cells, we found an accumulation of Cdc25C in the interphase, indicating that Cdc25C had not been translocated to the nucleus and so had not been phosphorylated (data not shown). Phosphorylated Cdc25C activates cdc2-cyclin B1 complex by dephosphorylating cdc2, resulting in an initiation of mitotic events. Our data clearly demonstrated that Plk1 depletion in PCa cells resulted in a significant up-regulation in the protein levels of total cdc2 and phosphorylated cdc2 (data not shown). Subsequent phosphorylation of cyclin B1 promotes the rapid nuclear translocation of cdc2-cyclin B1 at the G2/M transition, where cyclin B1 is then degraded. Our data showing an accumulation of cyclin B1 protein levels in Plk1 depleted cells (data not shown) further supported the hypothesis that the mechanism by which Plk1 depletion induces mitotic arrest is via the Cdc25C-cdc2-cyclin B1 feedback loop.

CONCLUSIONS

This in vitro study was an effort to investigate whether Plk1 could be exploited to design novel approaches to treat PCa, a growing concern worldwide, more so in developed countries. Plk1 is the most well-studied member of the Plk family of conserved serine-threonine kinases and has been shown to be a key regulator of mitotic progression by playing a critical role in events important for cell division. Recent studies have suggested that Plk1 could be a useful target for anti-tumor therapies, but an exact role of Plk1 in PCa is not well established. Our study demonstrated that Plk1 is overexpressed in human PCa cells and is undetectable in the PrEC cells and that the targeted depletion of Plk1 resulted in a significant decrease in growth and viability of all PCa cell lines studied without any effect on the normal PrEC, suggesting that Plk1 depletion could be useful in controlling the growth/development of PCa. Our data demonstrated that Plk1 depletion in PCa cells resulted in 1) an induction of apoptosis, 2) a mitotic cell cycle arrest, 3) failure of cytokinesis, 4) defects in centrosome integrity and maturation, and 5) accumulation of Cdc25C, cdc2, phospho-cdc2, and cyclin B1 proteins.

Taken together, our data support the hypothesis that Plk1 plays a critical role in PCa, and silencing of Plk1 will result in the elimination of human PCa cells via an inactivation of cyclin-dependent kinase 1 (Cdc2)/cyclin B1-mediated mitotic arrest, followed by apoptosis (Fig. 3 ). It is conceivable that gene therapeutic approaches aimed at Plk1 or pharmacological inhibitors aimed at Plk1 may be developed to manage PCa. Our data demonstrated that Plk1 depletion results in effective elimination of the three PCa cell lines observed, suggesting that Plk1 depletion-mediated mitotic arrest and apoptosis is a general phenomenon for PCa cells irrespective of 1) stage of PCa progression, 2) status of tumor suppressor p53 gene, and 3) association with androgen receptor. Thus, Plk1 inhibition-based approaches may be useful for the management of early to advanced stages of PCa.

View larger version (31K): [in this window] [in a new window]   Figure 3. Proposed mechanism of Plk1 depletion-mediated elimination of human PCa cells.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2910fje; doi: 10.1096/fj.04-2910fje

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