ARTICLE

Cancer Inhibition in Nude Mice After Systemic Application of U6 Promoter–Driven Short Hairpin RNAs Against PLK1

Birgit Spänkuch, Yves Matthess, Rainald Knecht, Brigitte Zimmer, Manfred Kaufmann, Klaus Strebhardt

Affiliation of authors: Department of Obstetrics and Gynecology, School of Medicine, J. W. Goethe-University, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany

Correspondence to: Professor Dr. K. Strebhardt, School of Medicine, 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)


    ABSTRACT
 Top
 Notes
 Abstract
 Introduction
 Materials And Methods
 Results
 Discussion
 References
 
Background: RNA interference initiated by small interfering RNAs effectively suppresses gene expression, but the suppression is transient, which limits the therapeutic use of this technique. Polo-like kinase 1 (PLK1) is a key cell cycle regulator that is overexpressed in various human tumors. We used a xenograft mouse model to determine whether an RNA interference–based strategy that used short hairpin RNAs (shRNAs) to suppress PLK1 expression could inhibit tumor growth in vivo. Methods: HeLa S3 cervical and A549 lung cancer cell lines were transfected with plasmids containing U6 promoter–driven shRNAs against human PLK1 or control (parental or scrambled) plasmids. Plasmids were treated with the nuclease inhibitor aurintricarboxylic acid (ATA) as protection against nucleases in murine blood. Nude mice carrying xenograft tumors were injected with shRNA plasmids, and their xenograft tumor growth was assessed. Northern and western blot analyses were used to measure PLK1 mRNA and protein expression, respectively, in transfected cultured cells and in xenograft tumors. All statistical tests were two-sided. Results: Levels of PLK1 mRNA and protein were lower in HeLa S3 and A549 cancer cells transfected with PLK1 shRNA plasmids than in corresponding cells transfected with control parental or scrambled PLK1S shRNA plasmids. Proliferation of cells transfected with PLK1 shRNA was lower than that of cells transfected with either control plasmid, and proliferation of cells transfected with ATA-treated PLK1 shRNA plasmids was even lower. In mice with human xenograft tumors, PLK1 shRNA expression from ATA-treated plasmids reduced tumor growth to 18% (95% confidence interval [CI] = 12% to 26%; P = .03) and from untreated plasmids reduced tumor growth to 45% (95% CI = 26% to 64%; P = .1) of that of tumors in mice treated with scrambled control PLK1S shRNA plasmids. Conclusions: The combination of shRNA-mediated gene silencing with effective in vivo gene delivery strategies appears to generate a long-lasting silencing signal.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials And Methods
 Results
 Discussion
 References
 
RNA interference is an excellent strategy for gene silencing (13). Tuschl and colleagues (4) showed that transfection of synthetic 21-nucleotide small interfering RNA (siRNA) duplexes into mammalian cells efficiently inhibits endogenous gene expression in a sequence-specific manner. However, phenotypic changes induced by siRNAs persist for at most 1 week, which limits their utility. Short hairpin RNAs (shRNAs) driven by polymerase III promoters have been investigated as an alternative strategy to more stably suppress gene expression, and such constructs with well-defined initiation and termination sites have been used to produce various small RNA species that inhibit the expression of genes with diverse functions in mammalian cell lines (511).

We have used RNA interference to investigate the role of the polo-like kinase 1 (PLK1) protein in neoplastic proliferation (12). PLK1 is a serine/threonine kinase that is highly conserved between yeasts and humans and plays an important role in cell cycle regulation (13). PLK1 expression is elevated in neoplastic tissues and may be a potential prognostic factor for many human cancers (14). All cancer cell lines (MCF-7 breast, HeLa S3 cervical, SW-480 colon, and A549 lung cancer cells) that we transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein compared with those levels in corresponding cells transfected with scrambled control siRNAs (12). Primary human mammary epithelial cells take up siRNAs less efficiently than do cancer cells, however, and transfection of such cells with PLK1 siRNAs slowed their proliferation only transiently (12).

In vivo delivery of siRNAs has been shown to inhibit transgene expression in certain organs of adult mice, predominately the liver (1518). The current knowledge about the inhibition of tumor cell proliferation by systemic treatment of tumor-bearing animals with siRNAs is very limited but is a potentially important therapeutic strategy because metastasis is the main cause of treatment failure and death from cancer. However, siRNAs without secondary modifications are unlikely to cause long-lasting changes. Consequently, we investigated whether transfection of cancer cells with plasmids expressing shRNAs targeted to human PLK1 and driven by a human U6 promoter inhibited the expression of PLK1 mRNA and protein and whether intravenous injection of such plasmids into mice carrying tumors would suppress tumor growth and PLK1 expression in tumor tissue.


    MATERIALS AND METHODS
 Top
 Notes
 Abstract
 Introduction
 Materials And Methods
 Results
 Discussion
 References
 
Plasmid Sequences

Plasmids were constructed by use of the pBS/U6 parental vector (10). To generate an intermediate plasmid for cloning shRNAs targeted to PLK1 (National Center for Biotechnology Information [NCBI] accession number X75932), the 21-nucleotide oligonucleotide 5'-GGCGGCTTTGCCAAGTGCTTA-3' annealed with the 25-nucleotide oligonucleotide 5'-AGCTTAAGCACTTGGCAAAGCCGCC-3' corresponding to siRNA2 (12) was inserted into parental plasmid pBS/U6 that had been digested with ApaI (blunted) and HindIII. The 21-nucleotide inverted motif, containing a 6-nucleotide spacer and a polymerase III termination signal of five thymidine residues (5'-AGCTTAAGCACTTGGCAAAGCCGCCCTTTTTG-3', 5'-AATTCAAAAAGGGCGGCTTTGCCAAGTGCTTA-3'), was subcloned into the HindIII and EcoRI sites of the intermediate plasmid to generate pBS/U6/shRNA/PLK1, hereafter termed the PLK1 shRNA plasmid. The same protocol was used to generate pBS/U6/shRNA/PLK1S (an shRNA in which the sequence targeted against PLK1 was randomized or scrambled), hereafter termed the control scrambled PLK1S shRNA plasmid.

Plasmids and Reagents

PLK1 shRNA, PLK1S shRNA, and parental plasmids were produced by PlasmidFactory (Bielefeld, Germany) as endotoxin-free research-grade quality. In brief, plasmid DNA was manufactured in two major phases. In the first phase, well-characterized Escherichia coli DH5{alpha} host cells were transformed with fully characterized plasmids. The resulting transformed bacteria were checked carefully for the expected characteristics (19). In the second phase, plasmid-producing cells were transferred to cultivation. This process requires the generation of a cell bank, which is essential for reproducible large-scale cultivation of bacterial biomass. Transformed bacteria were cultured without antibiotics, to avoid having to demonstrate that the purified plasmid DNA is free of contaminating antibiotics, and without animal-derived substances.

The bacterial biomass was subjected to quality control tests to verify that the product was present and that contaminating DNA was not present, and then plasmids were released by alkaline lysis. Subsequently, plasmids were isolated chromatographically from the soluble fraction of the lysate (19). More than 90% of the isolated plasmid DNA in such a preparation was covalently closed circular DNA. DNA concentration was adjusted to 1.0 mg/mL in phosphate-buffered saline (PBS), and the preparation was separated into 200-µL aliquots that were placed in DNA storage vials, frozen, and stored at –20 °C until use.

pPuro, a plasmid encoding the gene for puromycin resistance, and puromycin were obtained from Clontech (Heidelberg, Germany). Aurintricarboxylic acid (ATA) was obtained from Sigma-Aldrich (Taufkirchen, Germany).

Cell Culture, In Vitro Transfection With the Expression Plasmids, and PLK1 Depletion

Ham’s F12 medium and fetal calf serum were purchased from PAA Laboratories (Cölbe, Germany). PBS, glutamine, penicillin–streptomycin, and trypsin were from Invitrogen (Karlsruhe, Germany). The tumor cell line MCF-7 (breast) was obtained from DSMZ (Braunschweig, Germany), and the tumor cell line A549 (lung) was obtained from CLS (Heidelberg, Germany). Both lines were cultured according to supplier instructions.

HeLa S3 cells were transfected with plasmids by use of the GenePORTER 2 protocol (PEQLAB, Erlangen, Germany). One day before transfection, cultures were set up at a density of 2 x 105 cells per 10-cm2 dish. The amount of plasmid used for transfections was between 0.5 µg and 1.5 µg of plasmid per 10-cm2 dish. Control cells were transfected with the control scrambled PLK1S shRNA plasmids. For the transfection, cells were incubated for 4 hours at 37 °C in serum-free Ham’s F12 medium with GenePORTER 2 and the plasmids, and then the same volume of fresh culture medium containing 20% fetal calf serum was added.

For PLK1 depletion, cells were cotransfected with recombinant plasmids (parental plasmid, PLK1 shRNA plasmid, and scrambled PLK1S shRNA plasmid) and pPuro plasmids at a molecular ratio of 10 : 1. Twenty-four hours after transfection, the medium was changed, and puromycin at a concentration of 2 µg/mL was added to select transfection-positive cells. Floating cells were removed after 2 days of drug selection, and the remaining cells were again cultured with puromycin at 2 µg/mL until further analysis (72 or 96 hours after the beginning of transfection).

Cells were harvested 72 or 96 hours after the beginning of transfection for northern blot analysis, western blot analysis, and immunofluorescence. The growth rate of 2 x 105 cells was determined by counting cells 48, 72, and 96 hours after the beginning of the transfection. All transfections were performed in triplicate.

Human Cancer Xenograft Model and Plasmid Treatment

Human cancer xenograft models were established as described previously (23). Briefly, HeLa S3 or A549 cells, respectively, were harvested, washed with PBS, and resuspended in normal culture medium, and then 2 x 106 cells were injected subcutaneously into the flank regions of athymic nude (nu/nu) NMRI (Naval Medical Research Institute) mice that were 8–10 weeks old. Tumors that developed were serially transplanted a minimum of three times in these mice. For the experiment, tumor fragments were implanted subcutaneously in both flanks of nude mice, which were then randomly assigned to three independent treatment groups of five mice (parental plasmid with ATA, PLK1 shRNA plasmid with ATA, PLK1 shRNA plasmid without ATA, and scrambled PLK1S plasmid with ATA). When a tumor reached a volume of 50–100 mm3, the mouse was subjected to systemic treatment with one of the following treatments: ATA-treated and untreated PLK1 shRNA plasmids, ATA-treated scrambled control PLK1S shRNA plasmids, and ATA-treated parental plasmids. Plasmids (10 µg), with or without 2 µg of ATA (DNA/ATA weight ratio = 5 : 1), in 500 µL of PBS were injected into the mouse tail vein three times a week (on Monday, Wednesday, and Friday). Tumor diameters were measured during the treatment period and for 4–6 weeks after the end of therapy, as described previously (23), and mean tumor diameters and their 95% confidence intervals (CIs) were calculated. On the day after the last treatment, animals of each treatment group were sacrificed, and their tumors were excised for northern blot, western blot, and immunohistochemical analyses.

Experiments with human cancer xenograft mouse models were approved by the Regierungspräsidium Darmstadt (AZ VI63-19C20/15-F1/17). All animal experiments were performed in certified laboratories of the School of Medicine in Frankfurt.

RNA Preparation and Northern Blots

We used northern blot analysis to assess the expression of PLK1 mRNA. Seventy-two hours after the beginning of transfection, total RNA was isolated by use of RNeasy mini-kits according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Probes for northern blots were generated by radiolabeling antisense PLK1 and {beta}-actin strands. Each reaction mixture contained 250 µCi of [{alpha}-32P]dCTP (6000 Ci/mmol) for each reaction, the other three deoxyribonucleoside 5'-triphosphates (dNTPs; each at 50 µM), and 10 pmol of either primer PLK1-17-low (5'-TGATGTTGGCACCTGCCTTCAGC-3', corresponding to positions 1533–1554 within the open reading frame of PLK1) or primer actin-2-low (5'-CATGAGGTAGTCAGTCAGGTC-3'), as described previously (12). The template used to generate probes corresponded to PLK1 codons 285–497. Northern blotting and hybridizations were carried out as described previously (12). All blots were reprobed with {beta}-actin probes as the loading control. PLK1 and {beta}-actin mRNA expression and ethidium bromide staining of 18S and 28S ribosomal RNA were quantified by use of a Kodak gel documentation system (model 1D 3.5). The ratio of PLK1 expression to {beta}-actin expression for in vitro experiments and the ratio of PLK1 expression to ethidium bromide staining for in vivo experiments were determined as described previously (12). Results are expressed as the percentage of the level in control cells transfected with scrambled control PLK1S shRNA plasmids for in vitro experiments and as the percentage of the level in mice treated with PLK1S shRNA plasmids for in vivo experiments.

Western Blot Analysis

The level of PLK1 protein expressed was determined by western blot analysis. Briefly, cells were lysed 96 hours after the beginning of transfection in lysis buffer containing 50 mM HEPES (pH 7.0), 250 mM NaCl, 0.1% Nonidet P-40, 10% (vol/vol) glycerol, 1 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride, and 0.01 nM Na3VO4, and the lysate’s protein concentration was determined as described previously (12). Alternatively, xenograft tumors were homogenized and then lysed in a buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% Tween 20, 1 mM dithiothreitol, 10 mM glycerol 2-phosphate, 1 mM NaF, and 0.1 mM Na3VO4. Total proteins (10–50 µg) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 12% gels and then transferred (at 85 V for 1.5 hours) to Immobilon-P membranes (Millipore, Bedford, MA). Membranes were incubated for 1 hour in PBS containing 5% powdered nonfat milk and monoclonal antibodies against PLK1 (diluted 1 : 250) and {beta}-actin (diluted 1 : 200 000) and then incubated for 30 minutes in PBS containing 5% nonfat dry milk and goat anti-mouse serum (diluted 1:2000) and visualized as described previously (12).

The expression of PLK1 protein and {beta}-actin protein was quantified by use of a Kodak gel documentation system (model 1D 3.5). To control for variation in loading, all PLK1 protein expression levels were first normalized to {beta}-actin protein expression levels. The resulting PLK1 protein levels were then presented as a percentage of PLK1 protein levels in cells or tumors treated with control scrambled PLK1S shRNA plasmids.

Determination of Cell Proliferation

The growth rate of 2 x 105 cells was determined by counting cells at 24, 48, 72, and 96 hours after the beginning of transfection. Cells were counted with a hemacytometer. Cell viability was assessed by using trypan blue staining. The number of control cells (incubated with the parental plasmid) after 96 hours was used as the reference for this calculation. The numbers of plasmid-treated (PLK1 shRNA plasmid and scrambled PLK1S shRNA plasmid) cells and parental plasmid–treated control cells were determined to obtain the percentage of proliferating cells.

Analysis of PLK1 Expression by Indirect Immunofluorescence

Indirect immunofluorescence of HeLa S3 cells was performed as described previously (20). Polyclonal rabbit anti-human PLK1 antibodies (21) were used at a 1 : 100 dilution. Monoclonal mouse {alpha}-tubulin antibodies (Sigma-Aldrich, Taufkirchen, Germany) were used at a 1 : 100 dilution. To visualize the PLK1 and {alpha}-tubulin antibodies, we used Cy3-conjugated donkey anti-rabbit immunoglobulin G (IgG) and dichlorotriazinylaminofluorescein (DTAF)-conjugated goat anti-mouse IgG (both heavy and light IgG chains; Dianova, Hamburg, Germany) at a 1 : 500 dilution. DNA was stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (Sigma-Aldrich, Taufkirchen, Germany). Cells were examined with a fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of x100.

Isolation of Plasmid DNA From Murine Blood

The nuclease inhibitor ATA has been tested in a recent intravenous infusion study that was examining a model system in baboons for mammalian xenograft rejection (22). To determine whether ATA prolongs the half-life of plasmids in mammalian blood, we mixed parental plasmids with various concentrations of the nuclease inhibitor ATA and then incubated the mixture with murine blood. Specifically, blood samples were collected from the vena cava of nude mice under Enfluran anesthesia (Ethrane; Abbott, Wiesbaden, Germany), and 1 mL of blood was immediately mixed with 1.6 mg of EDTA. Plasmid DNA was mixed with ATA at a weight ratio of 50 : 1, 5 : 1, or 0.5 : 1 and incubated with blood from nude mice at 37 °C for 5 minutes to 4 hours. Samples were taken after 5 minutes, 30 minutes, 2 hours, and 4 hours. The stability of U6 promoter–containing plasmids was determined by isolating total DNA with QIAamp DNA Mini kits according to the manufacturer’s protocol (Qiagen) and then separating the plasmids by electrophoresis on 1% agarose gels or 1% TBE gels in Tris-buffered EDTA (1 mM EDTA and 10 mM Tris, pH 8.5).

Southern Blot Analysis for Stability of Plasmid DNA

To determine the stability of plasmids incubated in murine blood, plasmids were incubated in murine blood, and then total DNA was isolated from the reaction mixture and electrophoresed, as described above. To depurinize and denature DNA, gels containing DNA bands were incubated first for 15 minutes in 0.25 M HCl on a shaker to induce double-strand breaks, then for 30 minutes under denaturing conditions in a solution of 1.5 M NaCl and 0.5 M NaOH, and then for two 15-minute periods in a neutralizing solution containing 1.5 M NaCl, 0.5 M Tris–HCl (pH 7.2), and 1 mM EDTA (pH 8.0). DNA bands were then transferred to nylon membranes as described for northern blotting analysis (12). Membranes were dried at room temperature, and DNA was fixed to the membranes by a 5-minute exposure to UV radiation on a UV transilluminator.

The PLK1 shRNA plasmid was detected with an {alpha}-32P–labeled probe generated from antisense PLK1 strands in the following reaction mixture: 100 µCi–200 µCi of [{alpha}-32P]dCTP (6000 Ci/mmol), the three other dNTPs (each at 50 µM), and 10 pmol of primer PLK1–150-as (5'-GCAGCAGAGACTTAGGCACAA-3'; i.e., positions 310–330 in the open reading frame of PLK1), as described previously (12). Blots were prehybridized for 20 minutes at 68 °C in QuickHyb buffer (Stratagene, Amsterdam, The Netherlands) and hybridized in fresh QuickHyb buffer containing probes (106 cpm/mL) for 1 hour at 68 °C. Membranes were washed twice in 2x SSC (1x SSC = 0.15 M NaCl plus 15 mM trisodium citrate dihydrate) for 15 minutes at 36 °C and exposed to MP Hyperfilms (Amersham Pharmacia Biotech, Freiburg, Germany). Bands were quantified by use of a Kodak gel documentation system (model 1D 3.5).

Isolation of Plasmid DNA From Tumors and Polymerase Chain Reaction (PCR)–Based Detection of Plasmid DNA

To evaluate the transfection efficiency in xenograft experiments, we isolated total DNA from tumors on the day after the last treatment by use of QIAamp DNA Mini kits, according to the manufacturer’s protocol (Qiagen, Hilden, Germany). Briefly, tumor lysates were centrifuged (at 16.1g at room temperature, 10 seconds) and filtered. In the last step, plasmids adsorbed to the membrane were eluted with TE buffer (10 mM Tris at pH 8.5 and 1 mM EDTA). Plasmids were detected by PCR with plasmid-specific primers pBS-500s (5'-GAATAGACCGAGATAGGGTTGAGT-3') and pBS-500as (5'-CGTCGTTTTACAACGTCGTGACTG-3'). PCR products were separated by electrophoresis in 1% agarose gels and stained with ethidium bromide. Bands were visualized on a UV transilluminator and quantified by use of a Kodak gel documentation system (model 1D 3.5).

Evaluation of Toxicology and Immunohistochemistry

Frozen tumor sections were stained with hematoxylin. Toxic reactions were evaluated by two independent pathologists who inspected serial organ sections for histological abnormalities.

Immunohistochemical analysis of xenograft tumors was performed as described previously (21). Slides were incubated with rabbit anti-human Ki-67 polyclonal antibodies (DAKO, Hamburg, Germany) at a dilution of 1 : 80 or with PLK1 monoclonal antibodies (Transduction Laboratories, Heidelberg, Germany) at a dilution of 1 : 50. Primary antibodies were visualized by the alkaline phosphatase anti–alkaline phosphatase method (DAKO).

Statistical Methods

All experiments were performed in triplicate. Standardization and statistic analyses were performed as described previously (12). For two-way analysis of variance, all treatment groups were compared with PLK1S shRNA-treated mice (in vivo) or with PLK1S shRNA-treated cells (in vitro). For immunohistochemical comparison of anti–Ki-67 and anti–PLK1 antibody-stained cells between groups, Mann-Whitney U tests were used. All statistical tests were two-sided.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials And Methods
 Results
 Discussion
 References
 
PLK1 shRNA–Mediated Inhibition of PLK1 Expression

We generated DNA constructs (pBS/U6/shRNA/PLK1 and pBS/U6/shRNA/PLK1S, respectively) for the synthesis of the following shRNAs that correspond to the recently described siRNA2: PLK1 shRNA, which efficiently inhibits PLK1 expression in HeLa S3 cells, and PLK1S shRNA, the scrambled version of siRNA2, which does not inhibit PLK1 expression (12). Each construct produced an shRNA composed of two 21-nucleotide PLK1 sequences in an inverted orientation to each other separated by a 6-nucleotide spacer, and each construct also had a 3' RNA polymerase III termination signal sequence of five thymidine residues (Fig. 1).



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Fig. 1. Strategy for generating short hairpin RNA (shRNA) specific for polo-like kinase 1 (PLK1). An inverted repeat is inserted at position +1 of the U6 promoter (positions –315 to +1). The specific motif is 21 nucleotides (nt) long and corresponds to the coding region of the PLK1 gene. The two sequences forming the inverted repeat are separated by a 6-nt spacer. A transcription termination signal for RNA polymerase III containing five thymidine residues is attached to the 3' end of the inverted repeat. The transcribed RNA is predicted to fold back to form a shRNA. Restriction endonuclease sites are indicated. The parental plasmid, without the inverted repeat, is pBS/U6. DNA sequences for pBS/U6/shRNA/PLK1, the PLK1 shRNA plasmid that inhibits the expression of PLK1, and pBS/U6/shRNA/PLK1S, the scrambled control PLK1S shRNA that does not inhibit the expression of PLK1, are shown.

 
We first used northern blot analysis to investigate whether transfection of HeLa S3 cells with PLK1 shRNA or PLK1S shRNA constructs altered the level of PLK1 mRNA, compared with that in cells transfected with the control parental plasmid pBS/U6. After 72 hours of transfection, the level of PLK1 mRNA in cells expressing PLK1 shRNA was statistically significantly lower than that in cells expressing the control scrambled PLK1S shRNA or the parental vector, at all plasmid concentrations tested (PLK1 shRNA at 0.5 µg = 49.3% reduction in PLK1 mRNA level compared with that in both types of control cells, 95% CI = 34% to 100%, P = .04; 1.0 µg = 50% reduction, 95% CI = 45% to 100%, P = .04; and 1.5 µg = 48.3% reduction, 95% CI = 44% to 100%, P = .03) (Fig. 2A).



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Fig. 2. Polo-like kinase 1 (PLK1)–specific short hairpin RNAs (shRNAs) and PLK1 expression in transfected cultured HeLa S3 cervical cancer cells. Cells were transfected with a combination of recombinant plasmids, as indicated, and pPuro, a plasmid carrying the gene for puromycin resistance (ratio = 1 pPuro/10 recombinant plasmids). A) Northern blot analysis of PLK1 mRNA expression. Seventy-two hours after transfection, PLK1 expression was examined by northern blot analysis. PLK1 expression was quantified by use of a Kodak gel documentation system (model 1D 3.5). To control for variability of loading and transfer, membranes were reprobed for {beta}-actin (primer actin-2-low (5'-CATGAGGTAGTCAGTCAGGTC-3'), and actin-normalized PLK1 mRNA levels were expressed as a percentage of levels in cells transfected with 1.5 µg of scrambled control PLK1S shRNA plasmids. B) Western blot analysis. Ninety-six hours after transfection, PLK1 protein expression was examined by western blot analysis using monoclonal anti-PLK1 antibodies diluted 1 : 250. To control for variability of loading, membranes were reprobed with monoclonal antibodies against {beta}-actin diluted 1 : 200 000. The expression of PLK1 protein and {beta}-actin protein was quantified by use of a Kodak gel documentation system (model 1D 3.5). To control for variation in loading, all PLK1 protein expression levels were first normalized to {beta}-actin protein expression levels. {beta}-Actin–normalized PLK1 protein levels were expressed as a percentage of PLK1 protein levels in cells transfected with scrambled control PLK1S shRNA plasmids. C) HeLa S3 cell proliferation. The growth rate of 2 x 105 cells was determined by counting cells at 24, 48, 72, and 96 hours after the beginning of the transfection period. Cells were counted with a hemacytometer. The number of control cells (incubated with the parental plasmid) after 96 hours was used as the reference. The percentage of surviving cells is given as a percentage of the number of cells transfected with PLK1S shRNA plasmids 96 hours after transfection (panels AC) Data are the mean of three independent experiments; error bars are the upper 95% confidence intervals. D) Immunofluorescence analysis of HeLa S3 cells transfected with scrambled control PLK1S shRNA plasmids (upper panel) or PLK1 shRNA plasmids (lower panel), respectively. Cells in telophase were analyzed 72 hours after transfection. DNA was localized with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride staining (blue); {alpha}-tubulin was localized by immunostaining with anti–{alpha}-tubulin antibodies and dichlorotriazinylaminofluorescein (DTAF)–conjugated goat anti-mouse immunoglobulin G (IgG) (green); PLK1 was localized by immunostaining with anti-PLK1 antibodies and Cy3-conjugated donkey anti–rabbit IgG (red).

 
We used western blot analysis to determine whether the reduced levels of PLK1 mRNA observed after PLK1 shRNA transfection also reflected reduced PLK1 protein expression (Fig. 2B). The level of PLK1 protein in cells transfected 96 hours earlier with PLK1 shRNA vectors was lower than that in cells transfected with the parental vector or the scrambled PLK1S shRNA vector at all plasmid concentrations tested (PLK1 shRNA at 0.5 µg reduced PLK1 protein levels, compared with that in both types of control cells, by 72% (95% CI = 40% to 100%; P = .03), 1 µg by 75% (95% CI = 57% to 100%;P = .01), and 1.5 µg by 69% (95% CI = 20% to 100%; P = .04).

We next determined whether cancer cell proliferation was affected by depleting cells of PLK1 by use of PLK1 shRNA vector transfection (Fig. 2C). Although the proliferation of cells transfected with the parental vector or the scrambled control PLK1S shRNA vector was not altered compared with untreated cells (data not shown), 96 hours after the end of puromycin selection, proliferation of HeLa S3 cells transfected with the PLK1 shRNA plasmid was reduced by 89% (95% CI = 71% to 100%; P = .04) with 1.5 µg of the PLK1 shRNA vector, by 77% (95% CI = 39% to 100%; P = .07) with 1.0 µg, and by 61% (95% CI = 6% to 100%; P = .1) with 0.5 µg, compared with the proliferation of cells transfected with the scrambled control PLK1S shRNA plasmid.

A previous immunofluorescence study (24) reported that PLK1 surrounds the chromosomes in prometaphase, appears as several discrete condensed bands along the spindle axis at the interzone in anaphase, and concentrates at the midbody during telophase and cytokinesis. Immunofluorescence staining of PLK1 shRNA-transfected HeLa S3 cells for {alpha}-tubulin and for PLK1 showed that the level of PLK1 protein was reduced in cells expressing PLK1 shRNA and that the midbody in telophase was only weakly stained with PLK1-specific antibodies (Fig. 2D). Reduced levels of PLK1 mRNA and protein were also detected with this technique in other tumor cell lines (A549 and MDA-MB-435) expressing PLK1 shRNA but not in these tumor lines expressing the control scrambled PLK1S shRNA (data not shown).

Nuclease Inhibitor ATA and Stability of Plasmid DNA in Mammalian Blood

A potential barrier to the successful transfection of foreign DNA into mammalian cells in vivo is the activity of various blood-borne nucleases. To determine whether ATA could protect plasmids from the nucleases in peripheral blood from nude mice, we tested ATA, which inhibits DNase I, RNase A, S1 nuclease, exonuclease III, and various endonucleases (22,25), in ex vivo plasmid degradation assays. In these assays, we kept the mass of DNA, the volume of peripheral blood, and the incubation temperature constant, but we varied the incubation time. Plasmid integrity was assessed by Southern blot analysis.

When pure plasmid DNA (PLK1 shRNA) was incubated in murine blood, most supercoiled plasmid had disappeared by 30 minutes, and the corresponding degradation products (circular and linear forms) were detectable for up to 4 hours (Fig. 3). If plasmids were first mixed with ATA at weight ratios of DNA to ATA of 50 : 1, 5 : 1, or 0.5 : 1 and then the mixture was added to murine blood, stability was higher for ATA-treated supercoiled DNA than for untreated supercoiled DNA (Fig. 3). Supercoiled DNA was still visible after 2 hours at a ratio of DNA to ATA of 0.5 : 1, but it was at the limit of detection with lower ATA concentrations, such as a ratio of 5 : 1 (Fig. 3C, indicated by arrows). Southern blot analysis revealed that the corresponding degradation products (circular and linear DNA) were detectable in murine blood for more than 4 hours (Fig. 3D). TBE gels resolve linear and circular DNAs better. After a 4-hour incubation and electrophoresis on TBE gels, the signal of circular DNA treated with the highest concentration of ATA was five times stronger than the signal of untreated circular DNA, indicating that ATA apparently protects plasmid DNA in mammalian blood, especially circular DNA (Fig. 3E).



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Fig. 3. Nuclease inhibitor aurintricarboxylic acid (ATA) and the stability of plasmid DNA in murine blood. One milliliter of blood from a nude mouse was incubated at 37 °C with plasmids and ATA at the indicated weight ratios for 5 minutes (A), 30 minutes (B), 2 hours (C), or 4 hours (D); total DNA was isolated from the reaction mixture, separated by electrophoresis on 1% agarose gels or 1% TBE gels, respectively, and transferred to nylon membranes. The integrity of the plasmid DNA was then examined by Southern blot analysis. The PLK1 shRNA plasmid was detected with {alpha}-32P–labeled probe generated from antisense PLK1 using the primer PLK1–150-as (5'-GCAGCAGAGACTTAGGCACAA-3'; i.e., positions 310–330 in the open reading frame of PLK1), as described previously (12). Control linearized (KpnI) and circular plasmids were also subjected to electrophoresis. Prolonged stability of supercoiled plasmid DNA is indicated by arrows (in panel C, DNA/ATA ratio = 5 : 1 and DNA/ATA = 0.5 : 1). E) DNA was separated on 1% TBE gels after 4 hours of incubation with ATA at the indicated ratios to separate the linear and the nicked circle forms better and was then examined by Southern blot analysis as described above. F) Human blood was incubated at 37 °C with plasmids and ATA for 4 hours at the indicated weight ratios. The stability of plasmids was analyzed by ethidium bromide staining of the gels. Bands were quantified by use of a Kodak gel documentation system (model 1D 3.5). Intensity of ethidium bromide–stained supercoiled plasmids with different DNA : ATA weight ratios (50 : 1, 5 : 1, and 0.5 : 1) was compared with the signal intensity of a defined quantity of supercoiled plasmid. L = linear DNA; C = circular DNA; S = supercoiled DNA.

 
When higher concentrations of ATA were added to plasmid DNA before incubation with mouse blood, the degradation of plasmid DNA decreased in all samples in a concentration-dependent manner. Addition of ATA also protected the integrity of U6 promoter–containing vectors in human blood: After a 4-hour incubation at 37 °C, the signal intensity of the supercoiled form increased from 75% at a ratio of DNA to ATA of 50 : 1 to 92% at a ratio of 0.5 : 1 compared with the signal intensity of a defined quantity of supercoiled plasmid (Fig. 3F). Thus, ATA protects plasmid DNA from degradation by nucleases in mammalian blood.

Antitumor Activity of PLK1 shRNA In Vivo

To evaluate whether PLK1 shRNA from ATA-treated plasmids inhibited PLK1 gene expression in vivo better than PLK1 shRNA from untreated plasmids, we treated nude mice carrying subcutaneously implanted tumor xenografts (HeLa S3 and A549 cells) of 50–100 mm3 with untreated plasmids or with ATA-treated plasmids. We administered plasmids (PLK1 shRNA, control scrambled PLK1S shRNA, or parental control plasmids, each at 0.33–0.4 mg/kg of body weight) in 0.5 mL of PBS with or without ATA treatment to mice by bolus intravenous injection via the tail vein three times a week for 26 days. Treatment with PLK1 shRNA plasmids statistically significantly reduced the growth of HeLa S3 tumors in mice compared with treatment with control scrambled PLK1S shRNA plasmids or parental plasmids (Fig. 4A). When PLK1 shRNA plasmids were mixed with ATA at a ratio of 5 : 1 and then the mixture was administered to tumor-bearing mice, ATA-treated plasmids more efficiently inhibited tumor growth than did untreated PLK1 shRNA plasmids. For the treatment period ending 42 days after transplantation, PLK1 shRNA expression from ATA-treated plasmids reduced tumor volume to 18% (95% CI = 12% to 26%;P = .03) of tumor volume from mice injected with the ATA-treated scrambled control vector PLK1S shRNA. By contrast, PLK1 shRNA expression from untreated plasmids reduced tumor growth to only 45% (95% CI = 26% to 64%; P = .1) of tumor volume from mice injected with the ATA-treated scrambled control plasmid. Thus, ATA treatment increased the inhibitory effect of PLK1 shRNA in the tumor xenografts. In addition, tumor growth (HeLa S3) did not resume during the first 4 weeks after treatment with ATA-treated PLK1 shRNA plasmids ended. Four weeks after the end of treatment, tumor volume in the group receiving ATA-treated PLK1 shRNA plasmids was reduced to 2.6% (95% CI = 1% to 4%; P = .005) of that in the group receiving ATA-treated control scrambled PLK1S shRNA plasmids. Tumor volume in the group receiving untreated PLK1 shRNA plasmids was reduced to 17% (95% CI = 7% to 28%;P = .04) of tumor volume of the group receiving ATA-treated control scrambled PLK1S shRNA plasmids.



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Fig. 4. Polo-like kinase 1 (PLK1)–specific short hairpin RNAs (shRNAs) driven by U6 promoters and the growth of HeLa S3 xenograft tumors and of A549 xenograft tumors in nude mice. HeLa S3 tumors (A) or A459 tumors (B) were transplanted subcutaneously into the flanks of nude mice. Plasmids and ATA at a ratio of 5 : 1 were administered to tumor-bearing mice by bolus intravenous injection 3 times a week (Monday, Wednesday, and Friday) for 26 days. Tumor diameter was measured with a caliper twice a week, and tumor volume was calculated by the formula V = {pi}/6 x Dl x Ds2, where V is volume, Dl is the largest diameter, and Ds is the smallest diameter. Data are expressed as the mean of all tumor volumes for each group; error bars are the upper 95% confidence intervals.

 
We observed no reduction in the body weight of mice treated with PLK1 shRNA or PLK1S shRNA plasmids (each at 0.33–0.4 mg/kg) with or without ATA treatment. We also found no histopathologic sign of adverse events in the heart, lung, liver, kidney, intestinum, brain, bone marrow, or lymphatic tissues after treatment with ATA-treated PLK1 shRNA or PLK1S shRNA plasmids. Specifically, we found no pericarditis, myocardial fibrosis, signs of muscular dysfunction, valvular abnormality, or conduction disturbance, which can be detected in the heart during radiotherapy or chemotherapy. The lung parenchyma was normal; we found no sign of pneumonitis, fibrosis, or inflammation. In the liver, we found no sign of inflammatory or regressive changes, such as fibrosis. In the kidneys, we found no evidence for tubulopathy, glomerulonephritis, or degenerative alterations. In the intestines, we found no sign of elevated cell death in the crypt epithelium, breakdown of the mucosal barrier, mucositis, or prominent compensatory or proliferative reaction. The brains appeared normal (no signs of vasculopathy or necrosis). In the bone marrow and lymphatic tissue, we found no altered proliferation and no immature cells.

We also investigated the antitumor activity of PLK1 shRNA plasmids in nude mice implanted subcutaneously with A549 tumor xenografts. Injection of A549 tumor–bearing mice with ATA-treated PLK1 shRNA plasmids at a weight ratio of 5 : 1 inhibited tumor growth to 9.8% (95% CI = 7% to 13%; P = .002) of tumor growth in mice receiving ATA-treated control PLK1S shRNA plasmids compared with a growth reduction to 18.5% (95% CI = 9% to 28%; P = .01) in mice receiving untreated PLK1 shRNA plasmid also compared with mice receiving ATA-treated control PLK1S shRNA plasmids (Fig. 4B). We observed some tumor growth in A549 tumor–bearing mice during the 6 weeks after injection with ATA-treated PLK1 shRNA plasmids was terminated: The volume of tumors in mice injected with ATA-treated PLK1 shRNA plasmids was 21% (95% CI = 3% to 39%; P = .007) of that in mice injected with control scrambled PLK1S shRNA plasmids with ATA. Tumors in mice injected with untreated PLK1 shRNA plasmids reached 42% (95% CI = 14% to 69%; P = .01) of that of tumors in mice injected with PLK1S shRNA plasmids with ATA. Thus, treatment of plasmids with ATA clearly enhanced the inhibitory effect of PLK1 shRNA in the HeLa S3 and A549 tumor xenografts without reducing the body weight of the mice.

Vector-Induced Decreased Expression of PLK1 and Antitumor Activity

To determine whether plasmid DNA was associated with the xenograft tumors, we isolated total DNA from HeLa S3 and A549 xenograft tumors and used PCRs to detect plasmid DNA. A 500-bp fragment was generated in PCR using plasmid (pBS/U6)-specific primers and tumor DNA from animals. We found that total tumor DNA from animals treated with the parental, PLK1 shRNA (with or without ATA) or PLK1S shRNA plasmids contained plasmid DNA, demonstrating that all plasmids could be found with xenograft tumor tissue in vivo (Fig. 5A, B).



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Fig. 5. Analysis of tumors excised after termination of short hairpin RNA (shRNA) therapy. Detection of plasmids in the HeLa S3 tumors (A) or in the A549 tumors (B) of mice from each of the four treatment groups: aurintricarboxylic acid (ATA)–treated parental plasmids, ATA-treated polo-like kinase 1 (PLK1) shRNA plasmids, untreated PLK1 shRNA plasmids, and ATA-treated scrambled control PLK1S shRNA plasmids. Primers against the parental plasmid were used as probes to detect all plasmids by polymerase chain reaction (PCR). Amplified products are shown after separation by electrophoresis on a 1% agarose gel. M = the DNA ladder. Levels of PLK1 mRNA were determined in HeLa S3 tumors (C) and in A549 tumors (D) after 26 days of plasmid treatment. Tumors were excised 27 days after the beginning of treatment (i.e., 1 day after the last injection of plasmid), total mRNA was isolated, and northern blot analysis was performed. To control for variability of loading, gels were stained with ethidium bromide before blotting, and PLK1 mRNA levels were normalized to the ethidium bromide–stained 18S and 28S ribosomal RNA bands. Data are expressed as the percentage of the PLK1 mRNA level in tumors treated with the scrambled control PLK1S shRNA plasmid; error bars are the upper 95% confidence intervals.

 
To evaluate the effect of PLK1 shRNA on PLK1 mRNA expression in tumor cells, we used northern blot analysis to measure PLK1 mRNA levels in total RNA that had been isolated from HeLa S3 xenograft tumors after a 26-day treatment with the parental, PLK1 shRNA, or scrambled PLK1S shRNA plasmids. Tumors of mice treated with PLK1 shRNA had lower levels of PLK1 mRNA than did tumors of mice treated with the parental or scrambled plasmids (Fig. 5C). PLK1 mRNA expression was lower in tumors of mice injected with ATA-treated PLK1 shRNA plasmids (25%, 95% CI = 2% to 60%; P = .007) than in mice injected with untreated PLK1 shRNA plasmids (28%, 95% CI = 6% to 64%; P = .02), both compared with PLK1 mRNA expression in tumors of mice injected with ATA-treated scrambled control PLK1S shRNA plasmids. Results with mice carrying A549 tumors were similar to results with HeLa S3 tumors. PLK1 mRNA expression was lower in mice with A549 tumors injected with ATA-treated PLK1 shRNA plasmids (30%, 95% CI = 0% to 78%; P = .02) than in such mice injected with untreated PLK1 shRNA plasmids (60%, 95% CI = 27% to 94%; P = .04), both compared with PLK1 mRNA expression in mice treated with ATA-treated scrambled control PLK1S shRNA plasmids (Fig. 5D).

To determine whether the reduced levels of PLK1 mRNA observed in HeLa S3 xenograft tumors treated with PLK1 shRNA reflect reduced PLK1 protein levels, we used western blot analysis (Fig. 6B). Injection of mice with ATA-treated PLK1 shRNA plasmids statistically significantly reduced the level of PLK1 protein in HeLa S3 tumors to 15% (95% CI = 0% to 49%; P = .004) of that detected in mice injected with ATA-treated control scrambled PLK1S shRNA plasmids, and injection of untreated PLK1 shRNA plasmids reduced the level of PLK1 protein to 24% (95% CI = 0% to 61%; P = .007) of that detected in mice injected with ATA-treated control scrambled PLK1S shRNA plasmids. As with PLK1 mRNA expression, results with mice carrying A549 tumors were similar to results with HeLa S3 tumors. Injection of mice with ATA-treated PLK1 shRNA plasmids statistically significantly reduced the level of PLK1 protein in A549 tumors to 29% (95% CI = 20% to 40%; P<.001) compared with that in mice treated with ATA-treated scrambled control PLK1S shRNA plasmids (data not shown).



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Fig. 6. Polo-like kinase 1 (PLK1) protein expression and cell proliferation in HeLa S3 xenograft tumors. Mice with xenograft tumors were treated with plasmids for 26 days. The four treatment groups were aurintricarboxylic acid (ATA)–treated parental plasmids, ATA-treated polo-like kinase 1 (PLK1) short hairpin RNA (shRNA) plasmids, untreated PLK1 shRNA plasmids, and ATA-treated scrambled control PLK1S shRNA plasmids. A) Immunohistochemical analysis of PLK1 levels (panels ad) and Ki-67 levels (panels eh) in HeLa S3 tumors from mice injected with ATA-treated PLK1 shRNA plasmids (a and e), ATA-treated scrambled control PLK1S shRNA plasmids (b and f), untreated PLK1 shRNA plasmids (c and g), or ATA-treated parental plasmid (d and h). B) PLK1 protein expression in HeLa S3 tumors after 26 days of plasmid treatment. Tumors were excised 27 days after the beginning of treatment (i.e., 1 day after the last injection of plasmid), total protein was isolated, and proteins were separated by sodium dodecyl sulfate–polyacrylamide electrophoresis on 12% gels, transferred to Immobilon-P membranes, and examined by western blot analysis with anti-PLK1 antibodies. To control for variability of loading, membranes were reprobed with antibodies against {beta}-actin. {beta}-Actin–normalized PLK1 protein levels were compared. Data are expressed as the percentage of PLK1 protein levels in tumors treated with the scrambled control PLK1S shRNA plasmid.

 
To further test the vector system in vivo, we used immunohistochemistry to measure the level of PLK1 gene expression in xenograft tumors. The percentage of PLK1-positive tumor cells was 0% (95% CI = 0% to 0%; P<.001) in mice injected with ATA-treated PLK1 shRNA plasmids and 22.5% (95% CI = 10% to 39%; P = .006) in mice injected with untreated plasmids (Fig. 6A, panels a and c, respectively). By contrast, 39.2% of tumor cells in mice injected with ATA-treated scrambled control PLK1S shRNA plasmids were PLK1 positive (Fig. 6A, panel b), and 31.3% of the tumor cells in mice injected with ATA-treated parental plasmids were PLK1 positive (Fig. 6A, panel d).

Cell proliferation in tumors from the various treatment groups was assessed immunohistochemically by use of Ki-67 antibodies (Fig. 6A). The percentage of Ki-67–positive cells in HeLa S3 xenograft tumors of mice injected with ATA-treated PLK1 shRNA plasmids was 0% (95% CI = 0% to 0%; P<.001; Fig. 6A, panel e), that in tumors of mice injected with untreated PLK1 shRNA plasmids was 27.3% (95% CI = 13% to 41%;P = .03; Fig. 6A, panel g), that in tumors of mice injected with ATA-treated scrambled control PLK1S shRNA plasmids was 44.8% (95% CI = 25% to 67%), and that in tumors with ATA-treated parental plasmids was 32.3% (95% CI = 18% to 55%) (Fig. 6A, panels f and h, respectively). Thus, immunostaining of tumors for Ki-67 and PLK1 indicated that the antineoplastic effects observed in tumors with ATA-treated PLK1 shRNA plasmids were associated with a marked inhibition of HeLa S3 tumor cell proliferation.


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials And Methods
 Results
 Discussion
 References
 
Although the feasibility and potential of siRNA in cancer therapy have not yet been demonstrated, siRNAs that have been chemically synthesized or inserted in plasmids have been shown to inhibit expression of transgenes, such as the gene for luciferase or the gene for green fluorescent protein, in adult mice (1518). Current experimental evidence that siRNAs can inhibit endogenous genes is limited to genes expressed in murine liver: The in vivo silencing effect of siRNA directed against the Fas receptor gene was tested for its potential to protect mice from liver failure and fibrosis in models of autoimmune hepatitis (26). After administration of Fas-specific antibodies that induce fulminant hepatitis, all untreated control mice died within 3 days, whereas 85% of mice pretreated with Fas siRNAs survived, suggesting that RNA interference can prevent disease in an animal model of autoimmune hepatitis. RNA interference was also used to inhibit production of hepatitis B virus replicative intermediates in cell culture and in immunocompetent and immunodeficient mice transfected with a hepatitis B virus plasmid (27). In another study, tail vein injection of adenovirus particles expressing murine-specific siRNAs against {beta}-glucuronidase reduced the activity of {beta}-glucuronidase in adult mice (18). However, systemically administered adenovirus vectors can provoke immune responses, and thus their effectiveness for peripheral gene transfer is limited (28). The consequences of inappropriate vector integration must also be considered: Despite the low integration efficiency, reports of retroviral mutagenesis in mice and in two human subjects have raised concern about the potential for recombinant adeno-associated virus–mediated insertion mutagenesis (2931).

By contrast, in vivo gene transfer of naked DNA is reproducible, simple, and safe, but degradation of the naked DNA by nucleases can be a problem. After numerous attempts in our laboratory, intravenous or intratumor injection of synthetic siRNA targeted to PLK1 failed to inhibit tumor growth in xenograft models (data not shown), probably because of the short half-life of PLK1-specific siRNAs. Within 15 minutes of incubation, the PLK1-specific siRNA and its scrambled counterpart were completely degraded in mammalian serum (data not shown). Stabilization of the siRNA by encapsulation in liposomes or coadministration with RNasin was not sufficient, because these protected siRNAs did not inhibit MCF-7 or SW-480 tumor growth in nude mice. Consequently, we investigated whether shRNA vectors were more stable than siRNA in mouse serum and thus could suppress tumor growth in nude mice.

We found that systemic administration of plasmid DNA carrying shRNA targeted to PLK1, even in the absence of nuclease inhibitors, reached the tumor and inhibited tumor growth. We found, when we treated plasmids with the nuclease inhibitor ATA and then injected the plasmids into mice, that even more plasmid DNA reached the tumor. This observation is consistent with those demonstrating that DNA transfection of macaque, murine, and human respiratory tissue can be enhanced by treating the DNA with ATA before administration (32). Information about the systemic application of ATA is limited, coming primarily from a study that investigated the effect of intravenous infusion of ATA on platelet aggregation in baboons (33). Although baboons receiving a daily dose of 24 mg of ATA per kilogram of body weight showed decreased platelet aggregation and increased coagulation time, baboons receiving 12 mg/kg daily had normal blood parameters. We did not observe thrombotic disorders in our experiments, in which mice received a much lower dose of ATA (80 µg/kg of body weight) infused with plasmid DNA three times a week.

In conclusion, we demonstrate for the first time, to our knowledge, that U6 promoter–driven shRNAs targeted against PLK1 suppress tumor growth in mice when administered intravenously with the nuclease inhibitor ATA. The combination of shRNA-mediated gene silencing with effective in vivo gene delivery strategies appears to generate a long-lasting silencing signal. These results support further characterization of therapeutic systems using stable RNA interference technology.


    NOTES
 Top
 Notes
 Abstract
 Introduction
 Materials And Methods
 Results
 Discussion
 References
 
We are grateful to S. Kappel, E. Weith, and K. Frank for their excellent technical support. We also thank Barbara J. Rutledge, PhD, for editorial assistance.

Supported by grants from the DFG (STR/8-1), the Messer Stiftung, the Sander Stiftung, and the Dresdner Bank (Schleicher-, Paul und Ursula Klein-, Manja und Ernst Mordhorst-Stiftung).


    REFERENCES
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 Notes
 Abstract
 Introduction
 Materials And Methods
 Results
 Discussion
 References
 

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Manuscript received October 16, 2003; revised April 7, 2004; accepted April 20, 2004.


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