Affiliation of authors: B. Spänkuch-Schmitt, M. Kaufmann, K. Strebhardt (Department of Obstetrics and Gynecology, School of Medicine), J. Bereiter-Hahn (Department of Biology), J. W. Goethe-University, Frankfurt, Germany.
Correspondence to: Prof. Dr. K. Strebhardt, Department of Obstetrics and Gynecology, J. W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany (e-mail: Strebhardt{at}em.uni-frankfurt.de).
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ABSTRACT |
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INTRODUCTION |
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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 progressionin 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-2, 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.
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MATERIALS AND METHODS |
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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 178200 of the PLK1 open reading frame; siRNA3, to positions 362384; siRNA4, to positions 14161438; and siRNA5, to positions 15721594. 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 608630 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 -tubulin antibodies (Serotec/Biozol, Eching, Germany), mouse monoclonal
-tubulin antibodies (Dianova, Hamburg, Germany), mouse monoclonal
-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
Hams F12 and fetal calf serum (FCS) were purchased from PAA Laboratories (Cölbe, Germany). Dulbeccos 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 suppliers 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 x 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 x 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 x 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 manufacturers protocol (Qiagen, Hilden, Germany). Probes for northern blots were generated by radiolabeling antisense strands for PLK1 and -actin using 250 µCi of [
-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 15331554 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 285497 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% SDSpolyacrylamide gel and transferred to an ImmobilonTM-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 0.51 µg of substrate [the cytoplasmic retention signal within human cyclin B1 (5,6)] and with 2 µCi of [-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 SDSpolyacrylamide (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 -tubulin (to visualize the spindle apparatus),
-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
-tubulin and monoclonal
-tubulin, or polyclonal rabbit PLK1 and monoclonal mouse
-tubulin. Cells were examined with a fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of x40 or with a confocal laser scanning microscope (Zeiss, Oberkochen, Germany) using a x100 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 manufacturers 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.
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RESULTS |
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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, AC, 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.
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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 siRNA25 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 siRNA25 led to a statistically significant reduction of PLK1 protein (Table 2
).
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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).
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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
-tubulin (Fig. 3, A
, upper panel). Staining for
-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).
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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 -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).
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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 (5.6566 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, CE
).
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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).
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DISCUSSION |
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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.
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NOTES |
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We are grateful to B. Hüls and O. Möbert for their excellent technical support. We thank Dr. H. Ackermann for statistical advice.
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Manuscript received January 30, 2002; revised September 17, 2002; accepted October 2, 2002.
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