Abstract

Background: Expression of polo-like kinase-1 (PLK1), which has several functions in mitotic progression, is elevated in a broad range of human tumors. To investigate the role of PLK1 in neoplastic proliferation, we used the technique of RNA interference. Methods: Cells from several different cancer cell lines (MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, and A549 lung cancer cells) were transfected with small interfering (si) RNAs targeted against the human PLK1 or lamin genes. Northern and western blot analyses were used to examine PLK1 gene expression in transfected cancer cells and normal cells (human mammary epithelial cells [HMECs]). The phenotype, proliferation, and cell cycle distribution of cells transfected with siRNAs were also monitored by fluorescence microscopy and fluorescence-activated cell sorting analysis. Results: All cancer cell lines transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein. siRNA4, which had the strongest inhibitory effect, reduced PLK1 mRNA in MCF-7 cells by 70% and PLK1 protein in MCF-7 cells by 95% 24 hours after transfection. Cell proliferation was reduced by between 66% and 99% 48 hours after transfection, and apoptosis was increased from 1%–5% to 13%–50% in transfected cells. Transfected SW-480 cells were mitotically arrested, and their centrosomes had lost the ability to nucleate microtubules. HMECs took up siRNAs less efficiently than cancer cells, and transfection with siRNAs targeted to PLK1 did not inhibit their proliferation. Conclusions: PLK1 function appears to be essential for centrosome-mediated microtubule events and, consequently, for spindle assembly. siRNAs targeted against human PLK1 may be valuable tools as antiproliferative agents that display activity against a broad spectrum of neoplastic cells at very low doses.

Increasing knowledge about the genetic control of cellular proliferation provides the basis for the rational design of specific therapeutic strategies aimed at the regulation of proliferative disorders such as cancer. Key regulators for the mitotic progression in mammalian cells are the polo-like kinases (PLKs), which are structurally related to the polo gene product of Drosophila melanogaster, Cdc5p of Saccharomyces cerevisiae, and plo1+ of Schizosaccharomyces pombe (1). The PLKs from yeast, insects, amphibians, and mammals are a group of highly conserved serine/threonine kinases, suggesting that the proteins have a close evolutionary and thereby functional relationship. PLK1, the mammalian PLK that is most closely related to Drosophila polo, is found predominantly at centrosomes and at the midbody of dividing cells.

The activity of PLK1 is elevated in tissues and cells with a high mitotic index, including cancer cells (2,3). An increasing body of evidence suggests that the level of PLK1 expression has prognostic value for predicting outcomes in patients with several different cancers, including non-small-cell lung cancer, squamous-cell carcinomas of the head and neck, melanomas, oropharyngeal carcinomas, and ovarian and endometrial carcinomas (4). The importance of PLK1 as a measure for the aggressiveness of a tumor seems to result from its different functions during mitotic progression—in particular, its role in the G2/M transition (phosphorylation of cyclin B1, a component of the mitosis-promoting factor) (57). PLK1 also phosphorylates substrates that are involved in several additional steps of mitotic progression, including components of the anaphase-promoting complex and components of the cytokinesis machinery (8,9).

To define the role of PLK1 in tumorigenesis more precisely, powerful tools are required to suppress its high-level expression in cancer cells. One such tool is RNA interference (RNAi). RNAi was originally detected in Caenorhabditis elegans as a biologic response to exogenous double-stranded RNA (dsRNA), which induces sequence-specific silencing of gene expression (10). dsRNA is much more effective than antisense RNA at inducing gene silencing (10). Further investigations revealed that RNAi can occur in many eukaryotic species (11,12). Additional studies (12,13) of the biochemical components of RNAi indicate the existence of a conserved machinery for dsRNA-induced gene silencing that acts in two steps. In the first step, an RNase III family nuclease called Dicer cuts the dsRNA into short (21- to 23-nucleotide) pieces called small interfering RNAs (siRNAs). The siRNAs then enter a multimeric nuclease complex that identifies target mRNAs through their homology to siRNAs and induces destruction of these mRNAs.

Mammalian cells have evolved a response to viral attacks that are accompanied by dsRNA representing replication intermediates. Key players of this antiviral response are dsRNA-activated protein kinase (11), which phosphorylates EIF-2α, thereby inducing a generalized inhibition of translation. Surprisingly, chemically synthesized siRNAs that mimic the products of Dicer activity can induce gene silencing in a broad spectrum of human and mouse cell lines without inducing a generalized response to dsRNA (14,15).

In this article, we report the use of RNAi to inhibit the expression of PLK1 in several different human cancer cell lines (MCF-7 breast cancer, HeLa S3 cervical cancer, SW-480 colon cancer, and A549 lung cancer) and in human mammary epithelial cells (HMECs) to determine the role of PLK1 in tumorigenesis. Northern and western blot analyses were used to determine whether transfection of cells with siRNAs targeted to PLK1 could suppress PLK1 function. Proliferation and apoptosis were also assayed in transfected cells.

Materials and Methods

siRNAs and Antibodies

siRNAs were synthesized by Dharmacon Research, Inc. (Lafayette, CO). Several siRNA sequences targeting PLK1 (National Center for Biotechnology Information [NCBI] accession number X75932) were synthesized: siRNA2 corresponds to positions 178–200 of the PLK1 open reading frame; siRNA3, to positions 362–384; siRNA4, to positions 1416–1438; and siRNA5, to positions 1572–1594. We also synthesized siRNA1, directed against the LMNA gene, which encodes two proteins of the nuclear lamina, lamins A and C (NCBI accession number X03444); siRNA1 corresponds to positions 608–630 relative to the start codon (14). A scrambled version of siRNA4, siRNA4S, was synthesized to use as a control. All siRNAs were 21 nucleotides long and contained symmetric 3′ overhangs of two deoxythymidines.

Monoclonal anti-human PLK1 antibodies for western blots were obtained from Transduction Laboratories (Heidelberg, Germany) and for immunoprecipitation were obtained from Zymed Laboratories (San Francisco, CA). Antibodies for immunofluorescence studies were rat polyclonal α-tubulin antibodies (Serotec/Biozol, Eching, Germany), mouse monoclonal α-tubulin antibodies (Dianova, Hamburg, Germany), mouse monoclonal γ-tubulin antibodies (Sigma-Aldrich, Taufkirchen, Germany), and rabbit anti-PLK1 polyclonal antibodies (16). For western blots, mouse monoclonal antibodies against lamins were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg), and mouse monoclonal antibodies against actin were obtained from Sigma-Aldrich. Goat anti-mouse secondary antibodies were obtained from Santa Cruz Biotechnology, Inc.

Cell Culture

Ham’s F12 and fetal calf serum (FCS) were purchased from PAA Laboratories (Cölbe, Germany). Dulbecco’s modified Eagle medium (DMEM), RPMI-1640, phosphate-buffered saline (PBS), Opti-MEM I, oligofectamine, glutamine, penicillin and streptomycin, and trypsin were obtained from Invitrogen (Karlsruhe, Germany). The tumor cell lines SW-480 (colon), MCF-7 (breast), and HeLa S3 (cervix) were obtained from DSMZ (Braunschweig, Germany), and the tumor cell line A549 (lung) was obtained from CLS (Heidelberg). Mammary epithelial basal medium (MEBM), growth medium supplements (MEGM SingleQuots), and the HMEC system were obtained from Clonetics (Verviers, Belgium). All cell types were cultured according to the supplier’s instructions.

In Vitro Transfection With siRNAs

Cancer cells and HMECs were transfected with siRNAs using the oligofectamine protocol (Invitrogen). In brief, 1 day prior to transfection, cancer cells were seeded, without antibiotics, at 5 × 105 cells per 25-cm2 culture flask, corresponding to a density of 40%–50% at the time of transfection. Cancer cells were transfected with siRNAs at a concentration of 56 nM (except in dose dependence experiments, in which concentrations of siRNA1, siRNA4, and siRNA4S ranged from 0.56 nM to 566 nM). Control cells were incubated with Opti-MEM I alone without siRNA or oligofectamine. Cancer cells were incubated with siRNAs plus oligofectamine in Opti-MEM I or in Opti-MEM I alone (control cells) at 37 °C for 4 hours, at which point one-third of the transfection volume of fresh culture medium with threefold-concentrated FCS (30%) was added. Cells were harvested 6, 24, and 48 hours after the beginning of the transfection period for mRNA analysis and 48 hours after the beginning of the transfection period for protein expression, kinase assays, indirect immunofluorescence, and fluorescence-activated cell sorting (FACScan) analysis. All transfections were performed in triplicate for each time point. The growth rate of 5 × 105 cells was determined by counting cells at 24, 48, 72, and 96 hours after the beginning of the transfection period.

HMECs were transfected as described above for cancer cell lines, except that siRNA concentrations ranged from 566 nM to 2 μM because initial studies (data not shown) had demonstrated that concentrations sufficient to inhibit proliferation of cancer cells had no substantial effect on HMECs. For the determination of PLK1 mRNA and lamin protein levels and for immunofluorescence and FACScan analysis, the concentration of siRNAs was 2 μM. After the 4-hour transfection, one-third of the transfection volume of fresh culture medium (MEBM) with threefold growth supplements (SingleQuots) was added. HMECs were harvested for all analyses 48 hours after the beginning of the transfection period. The growth rate of 5 × 105 cells was determined by counting cells at 24, 48, 72, and 96 hours after the beginning of the transfection period.

RNA Preparation and Northern Blots

Total RNAs were isolated using RNeasy mini-kits, according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Probes for northern blots were generated by radiolabeling antisense strands for PLK1 and β-actin using 250 μCi of [α-32P]dCTP (6000 Ci/mmol) for each reaction, 50 μM of each of the other three dNTPs, and 10 pmol of either primer PLK1-17-low (5′-TGATGTTGGCACCTGCCTTCAGC-3′), corresponding to position 1533–1554 within the open reading frame of PLK1, or primer actin-2-low (5′-CATGAGGTAGTCAGT CAGGTC-3′), as described previously (17). The template for the generation of probes corresponds to amino acids 285–497 of PLK1. Northern blotting and hybridizations were carried out as described previously (17). All blots were reprobed with actin probes so that actin-normalized PLK1 mRNA levels could be compared. The normalized PLK1 mRNA levels are presented relative to those in siRNA4S-treated cells to control for transfection- or random siRNA-related effects.

Western Blot Analysis

For western blot analysis of cancer cells, cells were lysed and protein concentration was determined as described (18). Fifty micrograms of total protein was separated on a sodium dodecyl sulfate (SDS)–polyacrylamide (12%) gel and were then transferred (at 85 V for 1.5 hours) to ImmobilonTM-P membranes (Millipore, Bedford, MA). Membranes were incubated for 1 hour in 5% powdered nonfat milk in PBS with monoclonal antibodies against PLK1 (1 : 250) and actin (1 : 200 000) or with monoclonal antibodies against lamins (1 : 100) and actin (1 : 200 000). The membranes were then incubated for 30 minutes in 5% nonfat dry milk with goat anti-mouse serum (1 : 2000), and proteins were visualized as described previously (18).

For western blot analysis of HMECs, cells were rinsed with PBS, removed from culture flasks, spun down (245g, 10 minutes, 4 °C), and lysed in SDS buffer (4% SDS, 20% glycerol, 0.12 M Tris [pH 6.8]) containing a protease inhibitor mixture (Boehringer Mannheim, Mannheim, Germany). Lysates were immediately boiled for 10 minutes, and protein concentration was measured (19). One hundred micrograms of total protein was separated on a 12% SDS–polyacrylamide gel and transferred to an Immobilon™-P membrane. Membranes were incubated for 1 hour in 5% nonfat dry milk with monoclonal antibodies against PLK1 (1 : 50) and actin (1 : 200 000) or with monoclonal antibodies against lamins (1 : 100) and actin (1 : 200 000) and then for 30 minutes in 5% nonfat dry milk with goat anti-mouse serum (1 : 2000). Proteins were then visualized as described previously (18).

PLK1 protein expression levels were routinely normalized to actin protein expression levels. The resulting actin-normalized PLK1 protein levels are presented relative to actin-normalized levels in siRNA4S-treated cells.

In northern and western blotting experiments, PLK1 and actin expression was quantified with a Kodak gel documentation system (1D 3.5; Eastman Kodak, Rochester, NY). Integration of signal intensities from scanned autoradiographs was followed by quantitative comparison of PLK1 and actin expression. That is, for each treatment the ratio of PLK1 and actin signals was determined.

Kinase Assays

To assay PLK1 kinase activity, cells were lysed (18), and PLK1 was immunoprecipitated from lysates by using monoclonal PLK1 antibodies. For each immunoprecipitation, 800 μg of total protein (from the lysates of control cells, siRNA4-treated cells, and siRNA4S-treated cells) was incubated with 0.5 μg of antibody for 1 hour at 4 °C on a rotator. The immunoprecipitates were incubated with 0.5–1 μg of substrate [the cytoplasmic retention signal within human cyclin B1 (5,6)] and with 2 μCi of [γ-32P]-labeled adenosine triphosphate for 30 minutes at 37 °C in kinase buffer (20 mM HEPES [pH 7.4], 150 mM KCl, 10 mM MgCl2, 1 mM ethylene glycol-bis(2-aminoethylether)-N,N,N′, N′-tetraacetic acid (EGTA), 0.5 mM dithiothreitol, 5 mM NaF, 0.1 mM Na3VO4). Products from the kinase reactions were fractionated on SDS–polyacrylamide (12%) gels (Bio-Rad Laboratories, Munich, Germany), and phosphorylated substrate was visualized by autoradiography. After visualization, gels were stained with Coomassie blue to assay for equal loading of substrate. An equal amount of immunoprecipitates was subjected to western blot analysis to confirm equal loading of PLK1 protein in kinase reactions.

Autoradiographs and Coomassie blue-stained gels were scanned with a Kodak gel documentation system (1D 3.5; Eastman Kodak), and signal intensities were determined. Ratios of signal intensities of phosphorylated substrate to substrate loading were calculated and presented relative to ratios in untreated control cells.

Determination of Cell Proliferation

Cells were counted with a hemacytometer. Cell viability was assessed by trypan blue staining. The number of control cells (incubated with Opti-MEM I without oligofectamine or siRNA) after 96 hours was used as the reference. The numbers of siRNA-treated and control cells were determined to obtain the percentage of proliferating cells.

Analysis of Cell Structure by Indirect Immunofluorescence

Indirect immunofluorescence was carried out as described (20) for subcellular localization of α-tubulin (to visualize the spindle apparatus), γ-tubulin (to localize centrosomes), and PLK1. DNA was stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Sigma-Aldrich). The following antibodies, all at a 1 : 100 dilution, were used: polyclonal rat α-tubulin and monoclonal γ-tubulin, or polyclonal rabbit PLK1 and monoclonal mouse α-tubulin. Cells were examined with a fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of ×40 or with a confocal laser scanning microscope (Zeiss, Oberkochen, Germany) using a ×100 oil-immersion objective.

FACScan Analysis

Cell cycle distribution and apoptosis were analyzed using a FACScan apparatus (BD Biosciences, Heidelberg, Germany). For the determination of cell cycle distribution, cells were harvested, washed with PBS, and probed with CycleTESTTM PLUS DNA reagent kit (BD Biosciences), according to the manufacturer’s protocol. For each transfection (control and each siRNA), 30 000 cells were analyzed in triplicate. The percentage of cells in different cell cycle phases was calculated using ModFit LT for Mac (BD Biosciences). For the detection of apoptotic phenotypes, harvested cells were fixed with ice-cold 70% ethanol and treated for 20 minutes at 37 °C with RNase A at 5 μg/mL and propidium iodide at 50 μg/mL. Subsequent analyses of cell cycle distribution and apoptosis were performed using CELLQuest software (BD Biosciences).

To determine whether effects exerted by siRNAs in cell culture are influenced by transfection efficiency, we used FACScan analysis to determine the uptake of fluorescein-labeled siRNA4 into MCF-7 cells and HMECs 24 hours after transfection. The fluorescence of 10 000 cells was determined after subtracting the background fluorescence of control cells.

Statistical Methods

Each western blot experiment was performed three or four times. Northern blots were performed in triplicate. Means of normalized (i.e., to actin) signal intensities and 95% confidence intervals (CIs) were calculated. For the determination of proliferation, cell numbers were determined in triplicate at each time point, and means and 95% CIs were calculated. FACScan analyses were carried out three times for each cell type. Two-way analysis of variance (ANOVA) (GraphPad Prism; GraphPad Software, Inc., San Diego, CA) was performed to consider random effects of individual gels and different siRNA treatments. For two-way ANOVAs, all siRNA treatment groups were compared with siRNA4S-transfected cells. All P values are two-sided, and P<.05 was considered statistically significant.

Results

Specific Inhibition of PLK1 mRNA and Protein Expression by siRNAs

We first tested the ability of siRNAs to reduce the endogenous level of PLK1 mRNA in the MCF-7, HeLa S3, SW-480, and A549 cancer cell lines. Transfection of MCF-7 breast cancer cells in vitro with siRNA2, siRNA3, siRNA4, or siRNA5 at a concentration of 56 nM did not reduce PLK1 mRNA statistically significantly at 6 hours after transfection relative to the effect of siRNA4S but led to a statistically significant loss of PLK1 mRNA at 24 and 48 hours after the beginning of transfection, to 28%–75% of the levels in cells treated with siRNA4S (Fig. 1, A–C, and Table 1; in all analyses, levels of PLK1 mRNA were normalized to the levels of actin mRNA). Transfection with PLK1-targeted siRNAs reduced PLK1 mRNA levels in the other cancer cells at various time points as well (Table 1). In HeLa S3 cells, reductions similar to those in MCF-7 cells were seen at 24 and 48 hours. In SW-480 cells, the reduction of PLK1 mRNA occurred rapidly (within 6 hours) but was statistically significant at all three time points only for siRNA4 and siRNA5. A statistically significant reduction of PLK1 mRNA in A549 cells was found for several time points and siRNAs.

We also tested the effects of PLK1-targeted siRNAs on primary cells to determine whether reduction of PLK1 expression is also feasible in nontransformed cells. For primary cells we used HMECs. Concentrations of siRNAs (0.56–566 nM) suitable for reduction of PLK1 mRNA levels in cancer cells did not cause reduction of PLK1 mRNA levels in HMECs (data not shown). When siRNAs were used at 2 μM, a statistically significant reduction of PLK1 mRNA was detected at 48 hours with several siRNAs compared with levels in siRNA4S-treated cells (Fig. 1, D, and Table 1).

We next examined whether the siRNA-mediated decreases in PLK1 mRNA in cancer cells are accompanied by a reduction of PLK1 protein levels 48 hours after siRNA transfection. Just as MCF-7 cells that were transfected with siRNA2–5 showed a statistically significant reduction in PLK1 mRNA levels as compared with cells transfected with siRNA4S, they showed a statistically significant reduction in levels of the 68-kd PLK1 protein (Fig. 1, E, and Table 2). In HeLa S3, SW-480, and A549 cells as well, the reduction of PLK1 mRNA induced by transfection with siRNA2–5 led to a statistically significant reduction of PLK1 protein (Table 2).

Although we were able to evaluate PLK1 mRNA levels in siRNA-transfected HMECs, we were unable to test the impact of siRNAs on PLK1 protein expression in these cells because the level of PLK1 protein even in untreated HMECs was at the limit of detection. This result is in line with our previous observations showing that the level of PLK1 protein is very low in primary cells with weak proliferative activity (16).

We next investigated whether the inhibition of a gene such as that for lamins A and C, which is not expressed at higher levels in tumor cells than in normal cells, would nevertheless, like PLK1, show differential reduction in response to siRNA in MCF-7 cells and HMECs. When both cell types were transfected with siRNA1, which is targeted to the lamin A/C gene, lamin proteins disappeared completely by 48 hours (Fig. 2, A and B). However, the concentrations of siRNAs that were required to see these effects differed markedly: 56 nM siRNA1 conferred maximal reduction of lamin proteins in MCF-7 cells, whereas 2 μM siRNA1 was necessary in HMECs. Because siRNA-mediated reductions in the levels of both PLK1 and lamins required much higher concentrations of siRNA in HMECs than in MCF-7 cells, this effect is unlikely to be gene-specific. Instead, it is more likely that primary cells require elevated levels of siRNA for efficient knockdown of gene expression because of poorer transfection efficiency (see last paragraph of the “Results” section).

PLK1 mRNA and protein levels in cancer cells and HMECs were not influenced to a statistically significant extent by transfection with scrambled siRNA4S or by siRNA1 compared with those in untransfected control cells (Fig. 1 and Tables 1 and 2). In addition, treatment with PLK1-specific siRNAs did not statistically significantly reduce the level of lamin protein in cancer cells and HMECs (data not shown). Thus, the effects of siRNAs appear to be sequence-specific. Furthermore, several different concentrations of siRNA4, which had the most pronounced inhibitory effect in all cell lines tested, led to the reduction of PLK1 protein in MCF-7 cells (Fig. 1, F, and Table 3). Transfection with concentrations of 0.56, 5.6, and 56 nM led to a statistically significant reduction in PLK1 protein (Table 3). Elevating the concentration to 566 nM diminished the inhibitory potential of transfected siRNA4, possibly due to a reduced transfection efficiency at high siRNA concentrations (14).

To test whether the observed inhibitory effects could be improved by a combination of different siRNAs, we cotransfected MCF-7 cells with siRNA4 and siRNA2, siRNA3, or siRNA5. PLK1 protein expression was completely abolished in the cotransfected cells (data not shown). Thus, transfection of cells with two siRNAs, each at 28 nM, exerts a stronger inhibitory effect on PLK1 transcript levels than transfection of cells with one siRNA at 56 nM.

To determine whether the lower PLK1 protein levels in siRNA-transfected cells would be reflected in lower kinase activity, we determined the total kinase activity of immunoprecipitated PLK1 from MCF-7 cells that had been transfected 48 hours earlier with siRNA4 or siRNA4S. The cytoplasmic retention signal of cyclin B1 was used as the exogenous substrate (5,6). In siRNA4-transfected cells, phosphorylation of the substrate was reduced to 18% of the level in control cells (incubated with Opti-MEM I but without siRNA or oligofectamine) (Fig. 1, G). In contrast, siRNA4S did not substantially reduce total kinase activity of PLK1 relative to that in control cells. Thus, transfection of cancer cells with siRNAs that are associated with lowered expression of PLK1 specifically reduces PLK1 activity.

Abrogation of Spindle Formation Associated With Reduced Levels of PLK1 Protein

In a previous study (21), microinjection of antibodies to PLK1 induced abnormal distribution of condensed chromatin and monoastral microtubule arrays that were nucleated from duplicated but unseparated chromosomes. We therefore analyzed the morphology of siRNA-transfected cancer cells with a strong reduction in PLK1 expression. For these studies we mainly used siRNA4, which we had found to be the most powerful inhibitor in multiple cancer cell lines. Whereas control cells proceeded through mitosis, cells transfected 48 hours earlier with siRNA4 arrested at different mitotic stages, depending on the cell type. SW-480 cells did not enter prophase, as evidenced by the lack of the chromosome condensation that is typical of prophase in the nuclei of DAPI-stained cells (Fig. 3, A, upper panel). Many of the cells had separated centrosomes that had moved to opposite ends of the nucleus, as shown by staining for γ-tubulin (Fig. 3, A, upper panel). Staining for α-tubulin showed that the centrosomes were devoid of any microtubule connection (Fig. 3, A, upper panel). From the intensity of DAPI fluorescence, it appeared that the nuclei contained 4N DNA despite missing chromatin condensation. Thus, even though the centrosomes had undergone the normal prophase separation, the nuclei of siRNA4-transfected SW-480 cells seem to persist in G2 phase. In control SW-480 cells, by contrast, centrioles organized astral microtubules in early prophase, and chromosomes underwent condensation in the nucleus (Fig. 3, A, lower panel).

DAPI-stained cultures of MCF-7 cells transfected with siRNA4, unlike SW-480 cells transfected with siRNA4, displayed numerous apoptotic nuclei but no mitotic stages. The culture supernatant contained many mitotic cells that had obviously lost substrate adhesion. In addition, metaphase and telophase chromosomal arrangements were rarely identified, and more than 90% of all cells in mitosis were characterized by highly condensed, knoblike chromosomes that remained in an overall structure resembling the shape of a nucleus. Finally, only a few chromosomes left this ensemble by drifting away from the nuclear-like structure (Fig. 3, B). This phenotype indicates that the nuclear envelope had broken down but that no further mitotic spindle-related arrangement of chromosomes had occurred. Therefore, chromatid separation did not take place. γ-Tubulin was distributed throughout the cytoplasm of these cells, and no microtubules were seen (data not shown). The lack of microtubules could be a consequence of cell death after loss of adhesion.

Transfection of HMECs with siRNA4 at a concentration of 56 nM, which induced these severe morphologic changes in SW-480 and MCF-7 cancer cells, had no effect on the morphology of these primary cells (data not shown). Even when siRNA4 was used at a concentration of 2 μM, a level that decreases PLK1 mRNA in HMECs, we still detected no morphologic alterations in the HMECs (Fig. 3, C).

Double immunofluorescence staining of siRNA4-transfected MCF-7 cells and HMECs for α-tubulin and PLK1 revealed a marked reduction of PLK1 protein in both cell types. Whereas untransfected MCF-7 cells had many normal mitotic figures that were associated with high levels of PLK1 expression, siRNA4-transfected MCF-7 cells had only a few abnormal mitotic figures, and these were devoid of PLK1 protein (Fig. 3, D, lower panel). Because mitotic HMECs are rare, HMECs were examined in interphase. PLK1 protein expression was low but detectable in untransfected HMECs and was barely detectable in HMECs transfected with siRNA4 (at 2 μM) (Fig. 3, D, upper panel).

Cell Cycle Arrest and Apoptosis Associated With Reduced Levels of PLK1 Protein

We next used FACScan analysis to examine whether the mitotic changes observed in siRNA4-transfected MCF-7 and SW-480 cells were associated with arrest at particular stages of the cell cycle. Cancer cells showed a strong G2/M arrest 48 hours after transfection with 56 nM siRNA4: SW-480 cells showed a fivefold increase in the percentage of cells in G2/M relative to control cells; MCF-7 cells, a threefold increase; HeLa S3 cells, a fivefold increase; and A549 cells, a twofold increase (Fig. 4, left and middle panels). In contrast, HMECs showed weak G2/M arrest (increase of 32%) following transfection with 2 μM siRNA4 (Fig. 4). The effect of siRNA5 transfection on cell cycle distribution in the different cancer cell lines was similar to that of siRNA4 (Fig. 4); however, the effect of siRNA2 and siRNA3 transfection was variable, with MCF-7 and SW-480 showing only weak G2/M arrest (increase of 10%–40%) and HeLa S3 and A549 cells showing stronger arrest (comparable to that induced by siRNA4 transfection). No substantial change in cell cycle distribution was detected in cancer cells or HMECs transfected with siRNA1 or siRNA4S as compared with untransfected cells (Fig. 4, right panel).

DAPI staining revealed an elevated number of cells with apoptotic nuclei in cultures of siRNA4-transfected MCF-7 cells (56 nM) as compared with untransfected MCF-7 cells (Fig. 5, A, lower panel). By contrast, no cells with apoptotic nuclei were detectable in cultures of siRNA4-transfected HMECs (2 μM) (Fig. 5, A, upper panel). Confocal laser scanning microscopy analyses also revealed an elevated number of apoptotic cells with disintegrated nuclear membranes and condensed chromatin in cultures of siRNA4-transfected MCF-7 cells (Fig. 5, B). To study the increase in apoptosis induced by siRNA in more detail, we investigated whether reduced PLK1 levels are associated with apoptosis in all cancer cell lines by determining the increase of sub-2N DNA content (which indicates apoptosis) with FACScan analysis (data not shown). Whereas untransfected cells contained 1%–5% cells with sub-2N DNA, transfection with siRNA4 was associated with sub-2N DNA contents of 17% in SW-480 cells, 33% in MCF-7 cells, 50% in HeLa S3 cells, and 13% in A549 cells. In contrast, siRNA4 transfection of HMECs did not increase the number of cells with sub-2N DNA content.

Antiproliferative Effect in Cancer Cells In Vitro Associated With Reduced Levels of PLK1 Protein

We next examined the effect of siRNA transfection on cancer cell proliferation. MCF-7 cells transfected with one of the PLK1-targeted siRNAs showed statistically significant reductions in growth 96 hours after transfection, as compared with untransfected control cells (siRNA2: 83%, 95% CI = 27% to 138%; siRNA3: 81%, 95% CI = 49% to 113%; siRNA4: 97%, 95% CI = 82% to 111%; siRNA5: 89%, 95% CI = 68% to 112%) (Fig. 6, A). Inhibition of proliferation by these siRNAs was dose-dependent (Fig. 6, B). MCF-7 cells transfected with siRNA1 or with siRNA4S at any concentration tested grew at rates similar to oligofectamine-treated cells. By contrast, transfection of MCF-7 cells with increasing concentrations of siRNA4 (5.6–566 nM) led to almost complete cell death by 48 hours after transfection (Fig. 6, B). Transfection with siRNAs targeted to PLK1 also resulted in a statistically significant reduction of proliferative activity in the other cancer cell lines (SW-480 cells: siRNA2, 67%, 95% CI = 55% to 79%; siRNA3, 75%, 95% CI = 73% to 77%; siRNA4, 97%, 95% CI = 95% to 99%; siRNA5, 97%, 95% CI = 95% to 99%; HeLa S3 cells: siRNA2, 94%, 95% CI = 94% to 96%; siRNA3, 91%, 95% CI = 84% to 98%; siRNA4, 99%, 95% CI = 98% to 101%; siRNA5, 98%, 95% CI = 91% to 102%; A549 cells: siRNA2, 71%, 95% CI = 62% to 79%; siRNA3, 66%, 95% CI = 64% to 68%; siRNA4, 75%, 95% CI = 73% to 77%; siRNA5, 73%, 95% CI = 71% to 75%) (Fig. 6, C–E).

As in the other analyses, HMECs were much less sensitive than cancer cells to PLK1-targeted siRNAs. In MCF-7 cells, a growth reduction of 84% was seen 24 hours after transfection with siRNA4 at a concentration of 5.6 nM, whereas in HMECs a growth reduction of 78% required transfection with 2 μM siRNA4 (Fig. 6, F).

Differential Uptake of siRNAs in MCF-7 Cells Compared With Primary Human Mammary Epithelial Cells

Because several lines of evidence indicated that HMECs are less sensitive than cancer cells to siRNAs against PLK1, we investigated whether different transfection efficiencies could explain the differential sensitivity. To compare transfection efficiency of siRNA4 between cancer cell lines and HMECs, uptake of fluorescein-labeled siRNA4 was measured. A FACScan analysis of 10 000 cells revealed that, when siRNA4 was used at increasing concentrations (from 56 nM to 1000 nM), the transfection efficiency of MCF-7 cells did not change substantially (56 nM, 89.8% uptake; 100 nM, 89.4%; 150 nM, 89.3%; 1000 nM, 89.3%). In contrast, in HMECs a range of concentrations between 56 nM and 2 μM led to a substantial increase in uptake of fluorescein-labeled siRNA4, from 49.2% to 75.7% (56 nM, 49.2% uptake; 1000 nM, 71.3%; 2000 nM, 75.7%) (Fig. 7). These differences may be due to a difference in cell membrane permeability between cancer cells and primary cells (22,23).

Discussion

Since the discovery (11) that siRNAs can be used to regulate gene expression via elements of the RNAi machinery, we have sought to exploit this biologic mechanism for the regulation of desired target genes such as PLK1. In addition, we intended to evaluate the role of PLK1 in the proliferation of human cancer cells. Our study demonstrated that transfection of cancer cells with siRNAs targeted against human PLK1 reduced the level of PLK1 transcripts in cell culture. In several different cancer cell lines, siRNA4 exhibited a pronounced inhibitory effect on PLK1 expression and cell growth at a concentration of 5.6 nM. The effect disappeared if the siRNA concentration was reduced below 0.56 nM. One of the most attractive features of siRNA-based gene silencing is the potent inhibitory effect at low concentrations. In comparison, phosphorothioate antisense oligonucleotides display IC50 (inhibitory concentration, 50%) values between 100 and 500 nM (24,25). Thus, whereas phosphorothioate antisense oligonucleotides may have limitations as pharmacologic agents due to their potential toxicity, such a limitation seems less likely in the case of siRNA.

For siRNAs to be useful as pharmacologic agents, they also need to be specific for the gene of interest. Several lines of evidence suggest that the PLK1 siRNAs achieve this. First, within a small set of tested siRNAs targeted to different regions of human PLK1, only certain siRNAs (i.e., siRNA4 and siRNA5) had a potent silencing effect. Variations in the effectiveness of different PLK1-specific siRNAs in a particular cell line may be influenced by the ability of that cell line to form each RNAi silencing complex, which might be due to different accessibility of certain regions in the target mRNA. Second, neither a scrambled version of siRNA4 (siRNA4S) nor an siRNA targeted to lamins had much effect on the level of PLK1 expression.

All four cancer cell lines tested (MCF-7 breast, HeLa S3 cervical, SW-480 colon, and A549 lung cancer) were responsive to the antiproliferative effects of siRNA4, raising the possibility that PLK1 silencing might be useful for the treatment of tumors in future in vivo experiments. The potential therapeutic usefulness of siRNAs to PLK1 is supported by the observation that primary epithelial cells were not affected by siRNAs at concentrations that had a strong effect on cancer cells. A low transfection efficiency of primary cells may explain their lower sensitivity to siRNAs. Whatever the explanation, toxic side effects in normal cells exerted by siRNAs targeted to human PLK1 are less likely than side effects caused by phosphorothioate antisense oligonucleotides (24,25). In summary, our study strongly suggests that the inhibitory effect of siRNA4 on PLK1 expression and the biologic consequences that appear to result from these inhibitory effects in cell culture occur through an RNA-silencing mechanism.

In previous studies, adenoviral delivery of dominant negative forms of PLK1 led to the inhibition of PLK1 function (26). However, the treatment of cancer patients with recombinant adenoviral vectors still faces considerable limitations (27). Although current (i.e., second- and third-generation) adenoviral vectors have lower levels of toxicity and result in more prolonged gene expression in vivo than earlier versions of adenoviral vectors (28), an important limitation in the use of recombinant adenoviruses has been the difficulty in obtaining efficient gene transfer on a second administration of virus because of formation of neutralizing antibodies. Because short nucleic acids have no antigenic properties, it is unlikely that siRNAs would induce the formation of antibodies. The ability to use siRNA to selectively target proteins, such as PLK1, that are involved in tumorigenesis gives rise to the possibility that these novel agents could be used, not only as a new class of chemotherapeutic agents for the systemic treatment of cancer patients, but also to gain a better understanding of the critical molecular events responsible for initiating and maintaining the cancer phenotype.

In this context, our results raise intriguing questions about the role of PLK1 in cancer cells. Centrosomes play a critical role in generating genetic instability in cancer cells (29,30). They contribute to spindle abnormalities and disturbed chromosome segregation, which are often accompanied by profound alterations in key cellular functions, including regulation of apoptosis, control of cell cycle progression and cell cycle checkpoints, and cell growth regulation. Recent observations have shown that centrosomal abnormalities can be detected in early forms of human prostate cancer (31). Extra centrosomes in cancer cells might lead to chromosome missorting and damage, causing aneuploidy, which may induce the loss of tumor suppressor genes or activate oncogenes. Thus, it is possible that centrosomes are a driving force behind cancer formation, not a consequence of it (31).

Several studies (21,25,26,32) have tested the impact of interfering with PLK1 on the function of mammalian centrosomes. The analysis of HeLa cells microinjected with PLK1-specific antibodies revealed monoastral microtubule arrays that were nucleated from duplicated but unseparated centrosomes (21). However, the use of RNA silencing allowed us to separate centrosome division from microtubule anchoring: Centrosomes still divided and separated from each other in siRNA4-transfected SW-480 cells but without obvious microtubule interaction. If the pericentriolar matrix surrounding centrioles becomes dissolved in early prophase, centrosomes will not be maintained in close proximity. Lack of PLK1 after siRNA treatment prevents the formation of the microtubule nucleation complex required for aster and spindle formation. Thus, knockdown of PLK1 function may induce different mitotic phenotypes in various cancer cells because of diverse defects in checkpoint control. In addition, in a previous study (33), the level of PLK1 mRNA in cancer cells was associated with the extent of its interaction with heat shock protein 90. Thus, varying endogenous levels of PLK1 transcripts in cancer cells may also influence the outcome of siRNA treatment, thereby contributing to the resulting phenotype. Given the effects of siRNA4 that we observed in cultured cancer cells, future experiments examining the effects of siRNA targeted to human PLK1 against tumors in xenograft experiments are of obvious importance.

Table 1.

Effect of small interfering RNA (siRNA) transfection for varying times on polo-like kinase-1 (PLK1) mRNA levels in MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, A549 lung cancer cells, and human mammary epithelial cells (HMECs)*

Cell type Control† siRNA1 siRNA2 siRNA3 siRNA4 siRNA5 
*PLK1 mRNA levels were normalized to actin mRNA levels, and the amount of PLK1 mRNA expression remaining is given as a percentage of PLK1 mRNA levels in cells transfected with siRNA4S. PLK1 mRNA levels were determined by northern blot analysis. HMECs were transfected for 48 hours only. Cancer cells were transfected with 56 nM siRNA, and HMECs with 2 μM siRNA. siRNA1 is targeted against lamins, siRNA2–5 are targeted against PLK1, and siRNA4S is a scrambled version of siRNA4. For each siRNA transfection and time point, the mean of three independent experiments, the P value, and the 95% confidence interval (CI) is given. P values were determined by two-way analysis of variance. 
†Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). 
 6 h 
MCF-7 104% (95% CI = 88% to 119%) 86% (95% CI = 72% to 101%) 73% (95% CI = 30% to 116%) 98% (95% CI = 40% to 157%) 39% (95% CI = −28% to 106%) 92% (95% CI = 27% to 157%) 
 P = .4 P = .06 P = .1 P = .9 P = .06 P = .7 
HeLa S3 100% (95% CI = 6% to 193%) 88% (95% CI = −75% to 252%) 88% (95% CI = 19% to 158%) 91% (95% CI = −16% to 199%) 59% (95% CI = −38% to 156%) 95% (95% CI = 81% to 109%) 
 P = 1.0 P = .8 P = .5 P = .8 P = .2 P = .3 
SW-480 107% (95% CI = 81% to 132%) 75% (95% CI = 28% to 122%) 68% (95% CI = 1% to 136%) 65% (95% CI = 13% to 117%) 26% (95% CI = −9% to 61%) 37% (95% CI = −9% to 84%) 
 P = .4 P = .1 P = .2 P = .1 P = .01 P = .03 
A549 98% (95% CI = 79% to 116%) 87% (95% CI = 82% to 92%) 67% (95% CI = 65% to 69%) 81% (95% CI = 55% to 107%) 44% (95% CI = −33% to 121%) 41% (95% CI = 25% to 57%) 
 P = .6 P = .008 P = .0003 P = .09 P = .09 P = .004 
 24 h 
MCF-7 105% (95% CI = 33% to 177%) 94% (95% CI = 59% to 129%) 46% (95% CI = 29% to 62%) 41% (95% CI = 22% to 60%) 30% (95% CI = 2% to 58%) 28% (95% CI = −2% to 57%) 
 P = .8 P = .5 P = .005 P = .005 P = .008 P = .009 
HeLa S3 104% (95% CI = 81% to 128%) 115% (95% CI = 67% to 163%) 51% (95% CI = 46% to 57%) 46% (95% CI = 29% to 62%) 18% (95% CI = 9% to 27%) 28% (95% CI = 17% to 39%) 
 P = .5 P = .3 P = .0006 P = .005 P = .0006 P = .001 
SW-480 121% (95% CI = 101% to 142%) 93% (95% CI = 31% to 155%) 58% (95% CI = −25% to 142%) 47% (95% CI = −8% to 101%) 30% (95% CI = −5% to 65%) 30% (95% CI = 17% to 42%) 
 P = .05 P = .7 P = .2 P = .05 P = .01 P = .002 
A549 110% (95% CI = 108% to 112%) 86% (95% CI = 40% to 132%) 73% (95% CI = 56% to 90%) 86% (95% CI = −64% to 237%) 29% (95% CI = −25% to 82%) 38% (95% CI = −9% to 85%) 
 P = .003 P = .3 P = .02 P = .7 P = .03 P = .03 
 48 h 
MCF-7 93% (95% CI = 39% to 146%) 70% (95% CI = 36% to 104%) 67% (95% CI = −22% to 156%) 48% (95% CI = −10% to 105%) 44% (95% CI = 31% to 56%) 75% (95% CI = −20% to 171%) 
 P = .6 P = .06 P = .3 P = .06 P = .003 P = .4 
HeLa S3 125% (95% CI = 94% to 156%) 113% (95% CI = 39% to 188%) 41% (95% CI = −32% to 113%) 29% (95% CI = 3% to 55%) 29% (95% CI = 9% to 49%) 22% (95% CI = −5% to 40%) 
 P = .07 P = .5 P = .07 P = .007 P = .004 P = .003 
SW-480 112% (95% CI = 88% to 136%) 107% (95% CI = 79% to 136%) 87% (95% CI = 39% to 136%) 71% (95% CI = 4% to 138%) 32% (95% CI = 5% to 58%) 36% (95% CI = −16% to 89%) 
 P = .2 P = .4 P = .4 P = .2 P = .008 P = .04 
A549 132% (95% CI = 74% to 191%) 110% (95% CI = 104% to 117%) 43% (95% CI = −16% to 101%) 54% (95% CI = 52% to 56%) 49% (95% CI = 21% to 78%) 58% (95% CI = 19% to 96%) 
 P = .1 P = .02 P = .05 P = .0002 P = .02 P = .04 
HMEC 209% (95% CI = 122% to 296%) 144% (95% CI = −43% to 332%) 103% (95% CI = −65% to 272%) 46% (95% CI = 12% to 79%) 73% (95% CI = −16% to 91%) 70% (95% CI = 38% to 102%) 
 P = .03 P = .4 P = .9 P = .02 P = .04 P = .06 
Cell type Control† siRNA1 siRNA2 siRNA3 siRNA4 siRNA5 
*PLK1 mRNA levels were normalized to actin mRNA levels, and the amount of PLK1 mRNA expression remaining is given as a percentage of PLK1 mRNA levels in cells transfected with siRNA4S. PLK1 mRNA levels were determined by northern blot analysis. HMECs were transfected for 48 hours only. Cancer cells were transfected with 56 nM siRNA, and HMECs with 2 μM siRNA. siRNA1 is targeted against lamins, siRNA2–5 are targeted against PLK1, and siRNA4S is a scrambled version of siRNA4. For each siRNA transfection and time point, the mean of three independent experiments, the P value, and the 95% confidence interval (CI) is given. P values were determined by two-way analysis of variance. 
†Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). 
 6 h 
MCF-7 104% (95% CI = 88% to 119%) 86% (95% CI = 72% to 101%) 73% (95% CI = 30% to 116%) 98% (95% CI = 40% to 157%) 39% (95% CI = −28% to 106%) 92% (95% CI = 27% to 157%) 
 P = .4 P = .06 P = .1 P = .9 P = .06 P = .7 
HeLa S3 100% (95% CI = 6% to 193%) 88% (95% CI = −75% to 252%) 88% (95% CI = 19% to 158%) 91% (95% CI = −16% to 199%) 59% (95% CI = −38% to 156%) 95% (95% CI = 81% to 109%) 
 P = 1.0 P = .8 P = .5 P = .8 P = .2 P = .3 
SW-480 107% (95% CI = 81% to 132%) 75% (95% CI = 28% to 122%) 68% (95% CI = 1% to 136%) 65% (95% CI = 13% to 117%) 26% (95% CI = −9% to 61%) 37% (95% CI = −9% to 84%) 
 P = .4 P = .1 P = .2 P = .1 P = .01 P = .03 
A549 98% (95% CI = 79% to 116%) 87% (95% CI = 82% to 92%) 67% (95% CI = 65% to 69%) 81% (95% CI = 55% to 107%) 44% (95% CI = −33% to 121%) 41% (95% CI = 25% to 57%) 
 P = .6 P = .008 P = .0003 P = .09 P = .09 P = .004 
 24 h 
MCF-7 105% (95% CI = 33% to 177%) 94% (95% CI = 59% to 129%) 46% (95% CI = 29% to 62%) 41% (95% CI = 22% to 60%) 30% (95% CI = 2% to 58%) 28% (95% CI = −2% to 57%) 
 P = .8 P = .5 P = .005 P = .005 P = .008 P = .009 
HeLa S3 104% (95% CI = 81% to 128%) 115% (95% CI = 67% to 163%) 51% (95% CI = 46% to 57%) 46% (95% CI = 29% to 62%) 18% (95% CI = 9% to 27%) 28% (95% CI = 17% to 39%) 
 P = .5 P = .3 P = .0006 P = .005 P = .0006 P = .001 
SW-480 121% (95% CI = 101% to 142%) 93% (95% CI = 31% to 155%) 58% (95% CI = −25% to 142%) 47% (95% CI = −8% to 101%) 30% (95% CI = −5% to 65%) 30% (95% CI = 17% to 42%) 
 P = .05 P = .7 P = .2 P = .05 P = .01 P = .002 
A549 110% (95% CI = 108% to 112%) 86% (95% CI = 40% to 132%) 73% (95% CI = 56% to 90%) 86% (95% CI = −64% to 237%) 29% (95% CI = −25% to 82%) 38% (95% CI = −9% to 85%) 
 P = .003 P = .3 P = .02 P = .7 P = .03 P = .03 
 48 h 
MCF-7 93% (95% CI = 39% to 146%) 70% (95% CI = 36% to 104%) 67% (95% CI = −22% to 156%) 48% (95% CI = −10% to 105%) 44% (95% CI = 31% to 56%) 75% (95% CI = −20% to 171%) 
 P = .6 P = .06 P = .3 P = .06 P = .003 P = .4 
HeLa S3 125% (95% CI = 94% to 156%) 113% (95% CI = 39% to 188%) 41% (95% CI = −32% to 113%) 29% (95% CI = 3% to 55%) 29% (95% CI = 9% to 49%) 22% (95% CI = −5% to 40%) 
 P = .07 P = .5 P = .07 P = .007 P = .004 P = .003 
SW-480 112% (95% CI = 88% to 136%) 107% (95% CI = 79% to 136%) 87% (95% CI = 39% to 136%) 71% (95% CI = 4% to 138%) 32% (95% CI = 5% to 58%) 36% (95% CI = −16% to 89%) 
 P = .2 P = .4 P = .4 P = .2 P = .008 P = .04 
A549 132% (95% CI = 74% to 191%) 110% (95% CI = 104% to 117%) 43% (95% CI = −16% to 101%) 54% (95% CI = 52% to 56%) 49% (95% CI = 21% to 78%) 58% (95% CI = 19% to 96%) 
 P = .1 P = .02 P = .05 P = .0002 P = .02 P = .04 
HMEC 209% (95% CI = 122% to 296%) 144% (95% CI = −43% to 332%) 103% (95% CI = −65% to 272%) 46% (95% CI = 12% to 79%) 73% (95% CI = −16% to 91%) 70% (95% CI = 38% to 102%) 
 P = .03 P = .4 P = .9 P = .02 P = .04 P = .06 
Table 2.

Effect of small interfering RNA (siRNA) transfection on polo-like kinase-1 (PLK1) protein levels in MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW-480 colon cancer cells, and A549 lung cancer cells*

Cell type Control† siRNA1 siRNA2 siRNA3 siRNA4 siRNA5 
*Western blot analysis of PLK1 protein was carried out 48 hours after the beginning of transfection with 56 nM siRNA. siRNA1 is targeted to the lamin genes; siRNA2, siRNA3, siRNA4, and siRNA5 are targeted to different regions of the PLK1 gene; and siRNA4S is a scrambled version of siRNA4. PLK1 protein levels are given as a percentage of levels in cells transfected with siRNA4S (after standardization to actin protein levels). For each siRNA transfection and time point, the mean of three independent experiments, the P value, and the 95% confidence interval (CI) is given. P values were determined by two-way analysis of variance. 
†Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). 
MCF-7 84% (95% CI = 33% to 135%) 64% (95% CI = −28% to 156%) 11% (95% CI = −11% to 33%) 15% (95% CI = −1% to 31%) 5% (95% CI = −12% to 22%) 9% (95% CI = −15% to 33%) 
 P = .3 P = .2 P = .003 P = .002 P = .002 P = .004 
HeLa S3 113% (95% CI = 82% to 144%) 105% (95% CI = 53% to 157%) 30% (95% CI = −10% to 71%) 21% (95% CI = 7% to 35%) 40% (95% CI = −9% to 88%) 31% (95% CI = 7% to 55%) 
 P = .2 P = .7 P = .02 P = .002 P = .03 P = .007 
SW-480 111% (95% CI = 93% to 129%) 101% (95% CI = 55% to 147%) 41% (95% CI = −7% to 90%) 37% (95% CI = 21% to 54%) 21% (95% CI = −2% to 44%) 89% (95% CI = −19% to 36%) 
 P = .2 P = 1.0 P = .03 P = .003 P = .005 P = .005 
A549 115% (95% CI = 89% to 140%) 94% (95% CI = 27% to 161%) 54% (95% CI = −14% to 121%) 34% (95% CI = −18% to 86%) 17% (95% CI = −4% to 38%) 38% (95% CI = −6% to 82%) 
 P = .2 P = .8 P = .1 P = .03 P = .001 P = .02 
Cell type Control† siRNA1 siRNA2 siRNA3 siRNA4 siRNA5 
*Western blot analysis of PLK1 protein was carried out 48 hours after the beginning of transfection with 56 nM siRNA. siRNA1 is targeted to the lamin genes; siRNA2, siRNA3, siRNA4, and siRNA5 are targeted to different regions of the PLK1 gene; and siRNA4S is a scrambled version of siRNA4. PLK1 protein levels are given as a percentage of levels in cells transfected with siRNA4S (after standardization to actin protein levels). For each siRNA transfection and time point, the mean of three independent experiments, the P value, and the 95% confidence interval (CI) is given. P values were determined by two-way analysis of variance. 
†Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). 
MCF-7 84% (95% CI = 33% to 135%) 64% (95% CI = −28% to 156%) 11% (95% CI = −11% to 33%) 15% (95% CI = −1% to 31%) 5% (95% CI = −12% to 22%) 9% (95% CI = −15% to 33%) 
 P = .3 P = .2 P = .003 P = .002 P = .002 P = .004 
HeLa S3 113% (95% CI = 82% to 144%) 105% (95% CI = 53% to 157%) 30% (95% CI = −10% to 71%) 21% (95% CI = 7% to 35%) 40% (95% CI = −9% to 88%) 31% (95% CI = 7% to 55%) 
 P = .2 P = .7 P = .02 P = .002 P = .03 P = .007 
SW-480 111% (95% CI = 93% to 129%) 101% (95% CI = 55% to 147%) 41% (95% CI = −7% to 90%) 37% (95% CI = 21% to 54%) 21% (95% CI = −2% to 44%) 89% (95% CI = −19% to 36%) 
 P = .2 P = 1.0 P = .03 P = .003 P = .005 P = .005 
A549 115% (95% CI = 89% to 140%) 94% (95% CI = 27% to 161%) 54% (95% CI = −14% to 121%) 34% (95% CI = −18% to 86%) 17% (95% CI = −4% to 38%) 38% (95% CI = −6% to 82%) 
 P = .2 P = .8 P = .1 P = .03 P = .001 P = .02 
Table 3.

Effect of small interfering RNA (siRNA) transfection on polo-like kinase-1 (PLK1) protein levels in MCF-7 breast cancer cells*

siRNA (concentration) PLK1 protein level remaining† 
*Western blot analysis of PLK1 protein was carried out 48 hours after the beginning of transfection with the indicated concentrations of siRNA1 (targeted to lamins), siRNA4 (targeted to PLK1), and siRNA4S (scrambled version of siRNA4). CI = confidence interval. 
†PLK1 protein levels were standardized to actin protein levels and are given as a percentage of levels in untransfected control cells, incubated with Opti-MEM I alone (no siRNA or oligofectamine). For each siRNA concentration, the mean of three independent experiments, the 95% CI, and the P value are given. P values were determined by two-way analysis of variance. 
siRNA1 (0.56 nM100% (95% CI = 73% to 126%), P = 1.0 
siRNA1 (5.6 nM101% (95% CI = 84% to 118%), P = .9 
siRNA1 (56 nM 76% (95% CI = 39% to 112%), P = .1 
siRNA1 (566 nM 74% (95% CI = 26% to 121%), P = .2 
siRNA4 (0.56 nM 11% (95% CI = −12% to 33%), P = .001 
siRNA4 (5.6 nM 29% (95% CI = −33% to 91%), P = .04 
siRNA4 (56 nM 12% (95% CI = −11% to 34%), P = .001 
siRNA4 (566 nM 86% (95% CI = 1% to 172%), P = .6 
siRNA4S (0.56 nM109% (95% CI = 36% to 182%), P = .7 
siRNA4S (5.6 nM 76% (95% CI = 2% to 150%), P = .4 
siRNA4S (56 nM 78% (95% CI = −10% to 165%), P = .5 
siRNA4S (566 nM 93% (95% CI = 79% to 106%), P = .2 
siRNA (concentration) PLK1 protein level remaining† 
*Western blot analysis of PLK1 protein was carried out 48 hours after the beginning of transfection with the indicated concentrations of siRNA1 (targeted to lamins), siRNA4 (targeted to PLK1), and siRNA4S (scrambled version of siRNA4). CI = confidence interval. 
†PLK1 protein levels were standardized to actin protein levels and are given as a percentage of levels in untransfected control cells, incubated with Opti-MEM I alone (no siRNA or oligofectamine). For each siRNA concentration, the mean of three independent experiments, the 95% CI, and the P value are given. P values were determined by two-way analysis of variance. 
siRNA1 (0.56 nM100% (95% CI = 73% to 126%), P = 1.0 
siRNA1 (5.6 nM101% (95% CI = 84% to 118%), P = .9 
siRNA1 (56 nM 76% (95% CI = 39% to 112%), P = .1 
siRNA1 (566 nM 74% (95% CI = 26% to 121%), P = .2 
siRNA4 (0.56 nM 11% (95% CI = −12% to 33%), P = .001 
siRNA4 (5.6 nM 29% (95% CI = −33% to 91%), P = .04 
siRNA4 (56 nM 12% (95% CI = −11% to 34%), P = .001 
siRNA4 (566 nM 86% (95% CI = 1% to 172%), P = .6 
siRNA4S (0.56 nM109% (95% CI = 36% to 182%), P = .7 
siRNA4S (5.6 nM 76% (95% CI = 2% to 150%), P = .4 
siRNA4S (56 nM 78% (95% CI = −10% to 165%), P = .5 
siRNA4S (566 nM 93% (95% CI = 79% to 106%), P = .2 
Fig. 1.

Effect of small interfering RNA (siRNA) transfection on polo-like kinase-1 (PLK1) mRNA and protein levels in MCF-7 breast cancer cells and human mammary epithelial cells (HMECs). A–C) PLK1 mRNA was subject to northern blot analysis 6 (A), 24 (B), and 48 (C) hours after the beginning of transfection of MCF-7 breast cancer cells with 56 nM siRNA. To control for variability of loading and transfer, membranes were reprobed for human β-actin, and actin-normalized PLK1 mRNA levels were compared. The amount of PLK1 mRNA expression remaining is given as a percentage of PLK1 mRNA levels in cells transfected with siRNA4S. Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). Scatterplots show results of three independent experiments (three different symbols), their means (indicated as a horizontal bar), and 95% confidence intervals (CIs). D) PLK1 mRNA levels in HMECs were measured 48 hours after the beginning of transfection with 2 μM siRNAs. Normalization, standardization, and comparison with siRNA4S-transfected cells were as in A–C.Fig. 1. E) Western blot analysis of PLK1 protein was carried out 48 hours after the beginning of transfection of MCF-7 breast cancer cells with 56 nM siRNA. Actin served as a loading control. The amount of PLK1 protein remaining is given as a percentage of levels in cells transfected with siRNA4S after standardization to actin levels. Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). Scatterplot shows results of three independent experiments, their means, and 95% CIs. A–E) siRNA1 is targeted against lamins, siRNA2–5 are targeted against PLK1, and siRNA4S is a scrambled version of siRNA4. F) Western blot analysis of PLK1 protein expression in MCF-7 cells 48 hours after transfection with varying concentrations (0.56–566 nM) of siRNA1, siRNA4, and siRNA4S. In this particular analysis, remaining PLK1 protein levels were standardized to levels in untransfected control cells (i.e., MCF-7 cells incubated with Opti-MEM I alone without siRNA or oligofectamine). Scatterplot shows results of four independent experiments, their means, and 95% CIs. G) Kinase activity of immunoprecipitated PLK1 protein 48 hours after transfection of MCF-7 cells with 56 nM siRNA4, compared with control and siRNA4S-transfected cells. Kinase activity was visualized by phosphorylation of the substrate, cytoplasmic retention signal (CRS) (upper panel). Coomassie blue staining served as control for variability of loading of substrate CRS (middle panel). Western blot analysis of immunoprecipitated PLK1 demonstrates that equal amounts of PLK1 were subjected to kinase assays (lower panel). For quantification, signal intensities of phosphorylated substrate (kinase activity) were normalized to the amount of CRS loaded in each lane, and the kinase activity remaining after siRNA transfection was compared to that in untransfected control cells (incubated with Opti-MEM I alone, without siRNA or oligofectamine).

Effect of small interfering RNA (siRNA) transfection on polo-like kinase-1 (PLK1) mRNA and protein levels in MCF-7 breast cancer cells and human mammary epithelial cells (HMECs). A–C) PLK1 mRNA was subject to northern blot analysis 6 (A), 24 (B), and 48 (C) hours after the beginning of transfection of MCF-7 breast cancer cells with 56 nM siRNA. To control for variability of loading and transfer, membranes were reprobed for human β-actin, and actin-normalized PLK1 mRNA levels were compared. The amount of PLK1 mRNA expression remaining is given as a percentage of PLK1 mRNA levels in cells transfected with siRNA4S. Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). Scatterplots show results of three independent experiments (three different symbols), their means (indicated as a horizontal bar), and 95% confidence intervals (CIs). D) PLK1 mRNA levels in HMECs were measured 48 hours after the beginning of transfection with 2 μM siRNAs. Normalization, standardization, and comparison with siRNA4S-transfected cells were as in A–C.Fig. 1. E) Western blot analysis of PLK1 protein was carried out 48 hours after the beginning of transfection of MCF-7 breast cancer cells with 56 nM siRNA. Actin served as a loading control. The amount of PLK1 protein remaining is given as a percentage of levels in cells transfected with siRNA4S after standardization to actin levels. Control cells were incubated with Opti-MEM I alone (no siRNA or oligofectamine). Scatterplot shows results of three independent experiments, their means, and 95% CIs. A–E) siRNA1 is targeted against lamins, siRNA2–5 are targeted against PLK1, and siRNA4S is a scrambled version of siRNA4. F) Western blot analysis of PLK1 protein expression in MCF-7 cells 48 hours after transfection with varying concentrations (0.56–566 nM) of siRNA1, siRNA4, and siRNA4S. In this particular analysis, remaining PLK1 protein levels were standardized to levels in untransfected control cells (i.e., MCF-7 cells incubated with Opti-MEM I alone without siRNA or oligofectamine). Scatterplot shows results of four independent experiments, their means, and 95% CIs. G) Kinase activity of immunoprecipitated PLK1 protein 48 hours after transfection of MCF-7 cells with 56 nM siRNA4, compared with control and siRNA4S-transfected cells. Kinase activity was visualized by phosphorylation of the substrate, cytoplasmic retention signal (CRS) (upper panel). Coomassie blue staining served as control for variability of loading of substrate CRS (middle panel). Western blot analysis of immunoprecipitated PLK1 demonstrates that equal amounts of PLK1 were subjected to kinase assays (lower panel). For quantification, signal intensities of phosphorylated substrate (kinase activity) were normalized to the amount of CRS loaded in each lane, and the kinase activity remaining after siRNA transfection was compared to that in untransfected control cells (incubated with Opti-MEM I alone, without siRNA or oligofectamine).

Fig. 2.

Effect of small interfering RNA (siRNA) transfection on lamin protein levels in MCF-7 breast cancer cells and human mammary epithelial cells (HMECs). Reduction of lamin protein was determined 48 hours after the beginning of transfection with 56 nM siRNA1 (MCF-7 cells; A) and 2 μM siRNA1 (HMECs; B). Actin served as a control for equal loading. Percentage of remaining levels was calculated as a percentage of lamin protein levels in control cells (incubated with Opti-MEM I alone and without siRNA or oligofectamine) after standardization to actin levels. Two replicate experiments were performed, and means and 95% confidence intervals are indicated if the confidence intervals differed from zero.

Fig. 2.

Effect of small interfering RNA (siRNA) transfection on lamin protein levels in MCF-7 breast cancer cells and human mammary epithelial cells (HMECs). Reduction of lamin protein was determined 48 hours after the beginning of transfection with 56 nM siRNA1 (MCF-7 cells; A) and 2 μM siRNA1 (HMECs; B). Actin served as a control for equal loading. Percentage of remaining levels was calculated as a percentage of lamin protein levels in control cells (incubated with Opti-MEM I alone and without siRNA or oligofectamine) after standardization to actin levels. Two replicate experiments were performed, and means and 95% confidence intervals are indicated if the confidence intervals differed from zero.

Fig. 3.

Immunofluorescence analysis of SW-480 colon cancer cells, MCF-7 breast cancer cells, and human mammary epithelial cells (HMECs) transfected with small interfering RNA (siRNA). Cells were transfected with siRNA4 (SW-480, 56 nM; MCF-7, 56 nM; HMECs, 2 μM) and analyzed 48 hours later for DNA by 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining (blue), for γ-tubulin by immunostaining (green), for α-tubulin by immunostaining (red in panels A–C, green in panel D), and for PLK1 by immunostaining (red in panel D). A) siRNA4 transfection of SW-480 cells resulted in abrogated spindle formation at centrosomes (upper panel). Arrows point to a cell with centrosomes devoid of any microtubule connection. Untransfected cells (incubated with Opti-MEM I alone, with no siRNA or oligofectamine) show normal spindle formation (lower panel). B) Supernatant of MCF-7 cell cultures after siRNA4 transfection contained many mitotic cells that were characterized by highly condensed, knoblike chromosomes. Some chromosomes were located adjacent to the nucleus, as indicated by arrows. C) siRNA4-transfected HMECs displayed centrosomes that were able to organize microtubules (upper panel). These cells proceeded through mitosis with normal phenotype (middle panel). Phenotypes of control cells were similar to those of siRNA4-transfected cells. D) Both MCF-7 cells and HMECs showed reduction of PLK1 protein after siRNA4 transfection. Untransfected MCF-7 cells had normal mitotic phenotypes, whereas siRNA4-transfected MCF-7 cells showed impaired mitoses. In HMECs no mitotic cells could be found; therefore, PLK1 reduction is shown in interphase cells.

Fig. 3.

Immunofluorescence analysis of SW-480 colon cancer cells, MCF-7 breast cancer cells, and human mammary epithelial cells (HMECs) transfected with small interfering RNA (siRNA). Cells were transfected with siRNA4 (SW-480, 56 nM; MCF-7, 56 nM; HMECs, 2 μM) and analyzed 48 hours later for DNA by 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining (blue), for γ-tubulin by immunostaining (green), for α-tubulin by immunostaining (red in panels A–C, green in panel D), and for PLK1 by immunostaining (red in panel D). A) siRNA4 transfection of SW-480 cells resulted in abrogated spindle formation at centrosomes (upper panel). Arrows point to a cell with centrosomes devoid of any microtubule connection. Untransfected cells (incubated with Opti-MEM I alone, with no siRNA or oligofectamine) show normal spindle formation (lower panel). B) Supernatant of MCF-7 cell cultures after siRNA4 transfection contained many mitotic cells that were characterized by highly condensed, knoblike chromosomes. Some chromosomes were located adjacent to the nucleus, as indicated by arrows. C) siRNA4-transfected HMECs displayed centrosomes that were able to organize microtubules (upper panel). These cells proceeded through mitosis with normal phenotype (middle panel). Phenotypes of control cells were similar to those of siRNA4-transfected cells. D) Both MCF-7 cells and HMECs showed reduction of PLK1 protein after siRNA4 transfection. Untransfected MCF-7 cells had normal mitotic phenotypes, whereas siRNA4-transfected MCF-7 cells showed impaired mitoses. In HMECs no mitotic cells could be found; therefore, PLK1 reduction is shown in interphase cells.

Fig. 4.

Effect of transfection with small interfering RNA (siRNA) against PLK1 (siRNA4) on cell cycle distribution of cancer cells and human mammary epithelial cells (HMECs). FACScan analysis of SW-480 colon cancer, MCF-7 breast cancer, HeLa S3 cervical cancer, and A549 lung cancer cells 48 hours after transfection with 56 nM siRNA4 and in HMECs 48 hours after transfection with 2 μM siRNA4 (left panel). Controls were incubated with Opti-MEM I alone (no siRNA or oligofectamine). The intensity of fluorescence (which is proportional to the DNA content) is given on the x-axis, and the number of fluorescent cells is given on the y-axis. Graph is a representation of the effects of siRNA1–5 and siRNA4S on cell cycle distribution in cancer cell lines and HMECs (right panel). Bars show means and 95% confidence intervals of three independent experiments.

Fig. 4.

Effect of transfection with small interfering RNA (siRNA) against PLK1 (siRNA4) on cell cycle distribution of cancer cells and human mammary epithelial cells (HMECs). FACScan analysis of SW-480 colon cancer, MCF-7 breast cancer, HeLa S3 cervical cancer, and A549 lung cancer cells 48 hours after transfection with 56 nM siRNA4 and in HMECs 48 hours after transfection with 2 μM siRNA4 (left panel). Controls were incubated with Opti-MEM I alone (no siRNA or oligofectamine). The intensity of fluorescence (which is proportional to the DNA content) is given on the x-axis, and the number of fluorescent cells is given on the y-axis. Graph is a representation of the effects of siRNA1–5 and siRNA4S on cell cycle distribution in cancer cell lines and HMECs (right panel). Bars show means and 95% confidence intervals of three independent experiments.

Fig. 5.

Effect of transfection with small interfering RNA (siRNA) against PLK1 (siRNA4) on apoptosis of cancer cells and human mammary epithelial cells (HMECs). A) DAPI (2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) staining for DNA of HMECs and MCF-7 cells 48 hours after transfection with siRNA4 (56 nM for MCF-7 cells, and 2 μM for HMECs) reveals apoptotic phenotypes in MCF-7 cells (arrows). B) Confocal laser scanning microscope analysis revealed apoptotic cells with disintegrated nuclear membranes and condensed chromatin in this phase-contrast image of MCF-7 cells. White arrows indicate apoptotic nuclei, and yellow arrows indicate normal nuclei.

Fig. 5.

Effect of transfection with small interfering RNA (siRNA) against PLK1 (siRNA4) on apoptosis of cancer cells and human mammary epithelial cells (HMECs). A) DAPI (2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride) staining for DNA of HMECs and MCF-7 cells 48 hours after transfection with siRNA4 (56 nM for MCF-7 cells, and 2 μM for HMECs) reveals apoptotic phenotypes in MCF-7 cells (arrows). B) Confocal laser scanning microscope analysis revealed apoptotic cells with disintegrated nuclear membranes and condensed chromatin in this phase-contrast image of MCF-7 cells. White arrows indicate apoptotic nuclei, and yellow arrows indicate normal nuclei.

Fig. 6.

Effect of small interfering RNA (siRNA) transfection on proliferation of cancer cells and human mammary epithelial cells (HMECs). Percentage of surviving cells is given as a percentage of the number of control cells after 96 hours of incubation with Opti-MEM I alone (no siRNA or oligofectamine). A, C–E) Survival of MCF-7 breast cancer cells (A), SW-480 colon cancer cells (C), HeLa S3 cervical cancer cells (D), and A549 lung cancer cells (E) over a period of 96 hours after transfection. B and F) Proliferation of MCF-7 cells (B) and HMECs (F) at various times after transfection with various concentrations of siRNA1, siRNA4, and siRNA4S (5.6–566 nM for MCF-7 cells, 56 nM–2 μM for HMECs). Line graphs (A, C–E) represent means of three independent experiments. Bar graphs (B and F) represent means and 95% confidence intervals of three different experiments.

Fig. 6.

Effect of small interfering RNA (siRNA) transfection on proliferation of cancer cells and human mammary epithelial cells (HMECs). Percentage of surviving cells is given as a percentage of the number of control cells after 96 hours of incubation with Opti-MEM I alone (no siRNA or oligofectamine). A, C–E) Survival of MCF-7 breast cancer cells (A), SW-480 colon cancer cells (C), HeLa S3 cervical cancer cells (D), and A549 lung cancer cells (E) over a period of 96 hours after transfection. B and F) Proliferation of MCF-7 cells (B) and HMECs (F) at various times after transfection with various concentrations of siRNA1, siRNA4, and siRNA4S (5.6–566 nM for MCF-7 cells, 56 nM–2 μM for HMECs). Line graphs (A, C–E) represent means of three independent experiments. Bar graphs (B and F) represent means and 95% confidence intervals of three different experiments.

Fig. 7.

Uptake of fluorescein-labeled small interfering RNA4 (siRNA4) by MCF-7 breast cancer cells and human mammary epithelial cells (HMECs). Fluorescence-activated cell sorting (FACScan) analysis of control cells (i.e., incubated with Opti-MEM I alone, no siRNA or oligofectamine) and fluorescein-labeled siRNA4-transfected MCF-7 cells (left panel) and HMECs (right panel). The concentration of siRNA4 is indicated at the upper right of each graph. All transfections were carried out for 24 hours. M1 denotes the fluorescence of control cells (background) and M2 that of fluorescein-labeled siRNA4-containing cells. The intensity of fluorescence, representing the uptake of fluorescein-labeled siRNA4, is indicated on the x-axis; the number of fluorescent cells is given on the y-axis.

Fig. 7.

Uptake of fluorescein-labeled small interfering RNA4 (siRNA4) by MCF-7 breast cancer cells and human mammary epithelial cells (HMECs). Fluorescence-activated cell sorting (FACScan) analysis of control cells (i.e., incubated with Opti-MEM I alone, no siRNA or oligofectamine) and fluorescein-labeled siRNA4-transfected MCF-7 cells (left panel) and HMECs (right panel). The concentration of siRNA4 is indicated at the upper right of each graph. All transfections were carried out for 24 hours. M1 denotes the fluorescence of control cells (background) and M2 that of fluorescein-labeled siRNA4-containing cells. The intensity of fluorescence, representing the uptake of fluorescein-labeled siRNA4, is indicated on the x-axis; the number of fluorescent cells is given on the y-axis.

Supported in part by grant 10-1212-St 1 from the Deutsche Krebshilfe, grant STR/8-1 from the Deutsche Forschungsgemeinschaft, and by the Messer Stiftung, the Sander Stiftung, and the Dresdner Bank.

We are grateful to B. Hüls and O. Möbert for their excellent technical support. We thank Dr. H. Ackermann for statistical advice.

References

1
Glover DM, Hagan IM, Tavares AA. Polo-like kinases: a team that plays throughout mitosis.
Genes Dev
 
1998
;
12
:
3777
–87.
2
Golsteyn RM, Schultz SJ, Bartek J, Ziemiecki A, Ried T, Nigg EA. Cell cycle analysis and chromosomal localization of human Plk1, a putative homologue of the mitotic kinases Drosophila polo and Saccharomyces cerevisiae Cdc5.
J Cell Sci
 
1994
;
107
(Pt 6):
1509
–17.
3
Holtrich U, Wolf G, Brauninger A, Karn T, Bohme B, Rubsamen-Waigmann H, et al. Induction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumors.
Proc Natl Acad Sci U S A
 
1994
;
91
:
1736
–40.
4
Strebhardt K. PLK (polo-like kinase). In: Creighton TE, editor. Encyclopedia of molecular medicine. New York (NY): Wiley and Sons, Inc.; 2001. p. 2530–2.
5
Toyoshima-Morimoto F, Taniguchi E, Shinya N, Iwamatsu A, Nishida E. Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase.
Nature
 
2001
;
410
:
215
–20.
6
Yuan J, Eckerdt F, Bereiter-Hahn J, Kurunci-Csacsko E, Kaufmann M, Strebhardt K. Cooperative phosphorylation including the activity of polo-like kinase 1 regulates the subcellular localization of cyclin B1. Oncogene. In press 2002.
7
Qian YW, Erikson E, Taieb FE, Maller JL. The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes.
Mol Biol Cell
 
2001
;
12
:
1791
–9.
8
Shirayama M, Zachariae W, Ciosk R, Nasmyth K. The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae.
EMBO J
 
1998
;
17
:
1336
–49.
9
Ohkura H, Hagan IM, Glover DM. The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells.
Genes Dev
 
1995
;
9
:
1059
–73.
10
Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.
Nature
 
1998
;
391
:
806
–11.
11
Sharp PA. RNA interference–2001.
Genes Dev
 
2001
;
15
:
485
–90.
12
Hammond SM, Caudy AA, Hannon GJ. Post-transcriptional gene silencing by double-stranded RNA.
Nat Rev Genet
 
2001
;
2
:
110
–9.
13
Bernstein E, Denli AM, Hannon GJ. The rest is silence.
RNA
 
2001
;
7
:
1509
–21.
14
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.
Nature
 
2001
;
411
:
494
–8.
15
Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA. Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems.
Proc Natl Acad Sci U S A
 
2001
;
98
:
9742
–7.
16
Yuan J, Horlin A, Hock B, Stutte HJ, Rubsamen-Waigmann H, Strebhardt K. Polo-like kinase, a novel marker for cellular proliferation.
Am J Pathol
 
1997
;
150
:
1165
–72.
17
Wolf G, Elez R, Doermer A, Holtrich U, Ackermann H, Stutte HJ, et al. Prognostic significance of polo-like kinase (PLK) expression in non-small cell lung cancer.
Oncogene
 
1997
;
14
:
543
–9.
18
Bohme B, VandenBos T, Cerretti DP, Park LS, Holtrich U, Rubsamen-Waigmann H, et al. Cell-cell adhesion mediated by binding of membrane-anchored ligand LERK-2 to the EPH-related receptor human embryonal kinase 2 promotes tyrosine kinase activity.
J Biol Chem
 
1996
;
271
:
24747
–52.
19
Garbe J, Wong M, Wigington D, Yaswen P, Stampfer MR. Viral oncogenes accelerate conversion to immortality of cultured conditionally immortal human mammary epithelial cells.
Oncogene
 
1999
;
18
:
2169
–80.
20
Kauselmann G, Weiler M, Wulff P, Jessberger S, Konietzko U, Scafidi J, et al. The polo-like protein kinases Fnk and Snk associate with a Ca(2+)- and integrin-binding protein and are regulated dynamically with synaptic plasticity.
EMBO J
 
1999
;
18
:
5528
–39.
21
Lane HA, Nigg EA. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.
J Cell Biol
 
1996
;
135
:
1701
–13.
22
Mullin JM, Laughlin KV, Ginanni N, Marano CW, Clarke HM, Peralta SA. Increased tight junction permeability can result from protein kinase C activation/translocation and act as a tumor promotional event in epithelial cancers.
Ann N Y Acad Sci
 
2000
;
915
:
231
–6.
23
Caruso RA, Speciale G, Inferrera A, Rigoli L, Inferrera C. Ultrastructural observations on the microvasculature in advanced gastric carcinomas.
Histol Histopathol
 
2001
;
16
:
785
–92.
24
Tamm I, Dorken B, Hartmann G. Antisense therapy in oncology: new hope for an old idea?
Lancet
 
2001
;
358
:
489
–97.
25
Spankuch-Schmitt B, Wolf G, Solbach C, Loibl S, Knecht R, Stegmuller M, et al. Downregulation of human polo-like kinase activity by antisense oligonucleotides induces growth inhibition in cancer cells.
Oncogene
 
2002
;
21
:
3162
–71.
26
Cogswell JP, Brown CE, Bisi JE, Neill SD. Dominant-negative polo-like kinase 1 induces mitotic catastrophe independent of cdc25C function.
Cell Growth Differ
 
2000
;
11
:
615
–23.
27
Vorburger SA, Hunt KK. Adenoviral gene therapy.
Oncologist
 
2002
;
7
:
46
–59.
28
Schiedner G, Morral N, Parks RJ, Wu Y, Koopmans SC, Langston C, et al. Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity.
Nat Genet
 
1998
;
18
:
180
–3.
29
Doxsey SJ. Re-evaluating centrosome function.
Nat Rev Mol Cell Biol
 
2001
;
2
:
688
–98.
30
Brinkley BR. Managing the centrosome numbers game: from chaos to stability in cancer cell division.
Trends Cell Biol
 
2001
;
11
:
18
–21.
31
Pihan GA, Purohit A, Wallace J, Malhotra R, Liotta L, Doxsey SJ. Centrosome defects can account for cellular and genetic changes that characterize prostate cancer progression.
Cancer Res
 
2001
;
61
:
2212
–9.
32
do Carmo AM, Tavares A, Glover DM. Polo kinase and Asp are needed to promote the mitotic organizing activity of centrosomes.
Nat Cell Biol
 
2001
;
3
:
421
–4.
33
Simizu S, Osada H. Mutations in the Plk gene lead to instability of Plk protein in human tumour cell lines.
Nat Cell Biol
 
2000
;
2
:
852
–4.