Pharmaceutical composition for suppressing undesired gene expression
Description
The present invention is concerned with a possibility to suppress expression or undesired gene products, and especially with a pharmaceutical composition enabling such suppression of undesired gene products in the treatment or prevention of cancer diseases or associated with viral infections.
Reverse genetics, a technique that proceeds from genotype to phenotype by gene-manipulation techniques, is an efficient strategy to assess the function of a gene. RNA interference (RNAi) has become an excellent approach for the analysis of gene function in invertebrates and plants (1 -3). Fire and his colleagues demonstrated that in C. elegans the presence of just a few molecules of double -stranded RNA (dsRNA) was sufficient to reduce dramatically the expression of a gene that was homologues to the dsRNA (4). During this so-called process of RNA interference (RNAi) 21 -25-nucleotide (nt) long RNAs known as short -interfering RNAs (siRNAs) were detectable that were complementary to both strands of the silenced gene and that were processed from a long dsRNA precursor (5-10). Genetic data on silencing through siRNAs revealed the involvement of an enzyme called Dicer (11), which contains two RNase III motifs, an RNA helicase domain and a dsRNA-binding domain. This enzyme, seems to catalyse the processing of long dsRNA to 21 -23-nt siRNA products (9;12). Amazing observations by Tuschl and colleagues revealed recently that transfection of synthetic 21 -nt siRNA duplexes into mammalian cells efficiently inhibits endogenous gene expression in a sequence -specific manner (13). However, a significant disadvantage of siRNAs is that their effects are transient, with phenotypes generated by transfection with such RNAs persisting for 1 week at most. Thus, several groups have sought to expand the utility of RNAi in mammalian cells by devising techniques to
induce stable and heritable gene silencing. Different reports take advantage of RNA polymerase III promoters, which have well-defined initiation and termination sites producing various small RNA species. Beyond these, the U6 snRNA promoter and the H1 RNA promoter have been well characterized (14-16). A number of investigators was able to use this approach to demonstrate, in multiple mammalian cell lines, robust inhibition of several endogenous and exogenous genes of diverse functions (17 -22). In our recent study the technique of RNA interference was used to define the role of polo-like kinase 1 (PLK1) in neoplastic proliferation (23). Expression of the PLK1 gene coding for a serine/threonine kinase that is highly conserved from yeasts to humans, is elevated in a broad range of human tumors (24). The importance of PLK1 as measure for the aggressiveness of a tumor seems to result from its leading role for mitotic progression in particular control of the G2/M transition (25;26). Moreover, PLK1 triggers also additional steps during mitotic progression: activity of the anaphase promoting complex and cytokinesis (27-29). All cancer cell lines (MCF-7 breast cancer cells, HeLa S3 cervical cancer cells, SW -480 colon cancer cells, and A549 lung cancer cells) transfected with low doses of siRNAs targeted to PLK1 had greatly decreased levels of PLK1 mRNA and protein. Cell proliferation was reduced and apoptosis increased in siRNA -treated cells. Transfected SW-480 cells were mitotically arrested and their centrosomes had lost the ability to nucleate microtubules. Interestingly, HMECs (human mammary epithelial cells) took up siRNAs less efficiently than cancer cells, and transfection with siRNAs targeted to PLK1 did not inhibit their proliferation. Thus, 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.
In addition, cyclin B1 has also been shown to be of utmost importance for cell cycle regulation and tumor progression: The proper regulation of cyclin B1 , the regulatory subunit for Ser/Thr kinase cdc2 (cdkl ) is essential for the entry into mitosis. Increasing evidence demonstrates that cyclin B1 is involved in checkpoint control and that its deregulated expression could
contribute to the chromosomal instability observed in human cancer. Overexpression of cyclin B1 is observed in various carcinomas including oesophageal squamous cell carcinoma, laryngeal squamous cell carcinoma and colorectal carcinoma. Furthermore, overexpression of cyclin B1 indicates a poor prognosis and induces resistance to radiotherapy in head and neck squamous cell carcinoma. Taken together, these observations indicate that deregulation of cyclin B1 could be involved in neoplastic transformation and possibly in tumorigenesis. Thus, downregulation or depletion of cyclin B1 in tumor cells could be an attractive strategy for antiproliferative therapy.
In recent years small interfering RNA (siRNA) has become a specific and powerful tool to turn off the expression of target genes (11). In the present work we used siRNAs targeting cyclin B1 to reduce the expression of cyclin B1 , resulting in impaired MPF kinase activity, and analysed the impact of these siRNAs on cellular proliferation and apoptosis in tumor cell lines of different origins.
Considering the siRNA technology as a powerful strategy in reverse genetics, which exceeds the potential of antisense and Ribozyme approaches, it is tempting to apply this technology to 'knock -down' the expression of genes in living animals. Recent studies describe methods for in vivo delivery of siRNAs to organs of adult mice and demonstrate inhibition of gene expression in various organs (2;30-32). Still, systemic treatment of tumor-bearing animals with siRNAs has not been investigated previously, but is of utmost interest, because the main cause of treatment failure and death of cancer is metastasis.
It is also of great interest to develop methods that allow the inhibition of viral mechanisms of infection of cells and the formation of viral progeny and expansion of the infection to other cells. Especially for viruses which cause severe diseases there is a vital desire to develop possibilities to either prevent infection after exposition to the virus or to stop the progression of
the disease, thereby allowing the patient's immune system to defeat the virus efficiently.
It was therefore an object of the present invention to develop corresponding treatments and pharmaceuticals that allow for effective and selective suppression of gene expression in vivo and that avoid drawbacks of the state of the art. It was especially desired to develop methods that can easily be applied and show a prolonged effectiveness as compared to already known gene silencing methods. 0
This object of the present invention was solved by providing pharmaceutical compositions and their uses for suppression of undesired gene expression. A first subject of the present invention, therefore, is a pharmaceutical composition for suppressing gene expression comprising an effective 5 amount of
1) an siRNA expression system and
2) a nuclease inhibiting substance, wherein said siRNA expression system contains a) an RNA polymerase specific promoter sequence and under the o transcriptional control of said promoter sequence b) genetic information homologous to said gene to be suppressed, such genetic information being transcribed under suitable conditions and in the presence of an RNA polymerase into short interfering double stranded RNA. 5
The interfering RNA is preferably a siRNA, in particular a shRNA (hairpin) or a short antisense RNA (e.g. 15-30, in particluar 20-25 nt in length).
In the framework of the investigations leading to the present invention it was 0 surprisingly found that an effective and persistent gene silencing is possible by way of addition of nuclease inhibiting substances to the siRNA generating expression system. This nuclease inhibiting substances avoid the breakdown and removal of the expression vector containing the genetic
information for the siRNA. Thus the expression vector in the presence of RNA polymerases which are abundant in vivo, can constitutively express siRNAs targeted against the gene to be silenced for a sufficiently long time. It was found that application of the pharmaceutical composition of the present invention every other day was sufficient to suppress tumor growth and to allow immune systems to attack the tumors and thereby even reduce tumour size. Also it was observed that viral infections can be stopped or at least their effects be substantially reduced by administration of the present pharmaceutical composition.
The pharmaceutical composition of the present invention further showed no marked detrimental side effects to the treated person. To the contrary, the application can take place easily by for example intravenous injection of the composition.
The following investigations lead to the development of the present pharmaceutical compositions and their use in gene silencing:
Experimental introduction of siRNA duplexes 21 -nt in length with 2 to 3 nt 3' overhanging ends containing 5' phosphate and free 3' hydroxyl termini (10;13) into mammalian cells is now widely used to disrupt the activity of cellular genes homologous in sequence to the transfected siRNA. Previous studies in Drosophila and C. elegans demonstrated that hairpin dsRNA, which might be further processed by Dicer, silences also expression of a specific gene (33;34). Subsequent investigations took advantage of the human U6 snRNA promoter or the H1 promoter and its simple termination signal to express short hairpin RNAs (shRNAs) for efficient 'knock -down' of gene function. To generate shRNA targeted to PLK1 or cyclin B1 from a plasmid-based expression system, we used a RNA Pollll-based strategy to synthesize transcripts terminating at a run of 4-5 thymidine nucleotides. This procedure warrants shRNAs with defined ends. We wished to test the possibility whether small endogenously encoded hairpin RNAs might also
regulate human PLK1 or cyclin B1 in mammalian cells thereby suppressing proliferative activity as recently demonstrated for synthetic siRNAs targeted to PLK1 (23). Since our previous experiments showed that synthetical siRNA2 targeted to human PLK1 is most effective in suppressing PLK1 mRNA expression in HeLa S3 cells, we inserted corresponding annealed oligonucleotides that acted as templates for the synthesis of shRNAs into a plasmid containing the mouse U6 promoter that directs the synthesis of a Pollll-specific RNA transcript (18). The resulting hairpin RNA is composed of two complementary sequences 21 nt in length in an inverted orientation separated by a spacer of 6 nt in length (Fig. 1). To function as a termination signal for Pollll 5 thymidine nucleotides were hooked onto the 3'end. Transcription from the U6 promoter generate RNAs that are predicted to form hairpins as secondary structure with a 3' overhang of several thymidines. We used this approach to generate DNA templates for the synthesis of shRNAs corresponding to the recently used siRNA2 (shRNA/PLK1) and a scrambled version of siRNA2 (shRNA/PLK1S). The same approach was used to generate shRNAs targeted against cyclin B1.
Northern- and Western blots revealed that the U6-driven expression of hairpin RNAs targeted to PLK1 or to cyclin B1 is suitable to inhibit expression of the corresponding protein in HeLaS3 and A549 cells. .
Although siRNAs are effective tools for inhibition of gene function in mammalian cells, their suppressive effects are by definition of limited duration. Thus, strategies are required that could bypass such limitations and provide a tool for evoking stable suppression by plasmid -driven expression of siRNAs or hairpin RNAs in mammalian cells. Cellular nuclease activity is a potential barrier to the successful delivery of foreign DNA to and its function within mammalian cells. We tested the hypothesis that transfection in the presence of specific nuclease inhibitors can enhance the expression of exogenous gene products. We used aurin tricarboxylic acid (ATA) to enhance the expression of U6 -driven shRNAs in the human cell
lines HeLa S3 and A549. ATA has been shown previously to inhibit DNase I, RNase A, S1 nuclease, exonuclease III and various endonucleases (35;36). As shown by proton magnetic resonance spectroscopy the mechanism of action of ATA involves competition between the nucleic acid and the polymeric ATA for binding in the active site of a polypeptide such as nucleases (37).
Administration of synthetical siRNA or virus -mediated siRNA in adult mice decreased partially the expression of exogenous or endogenous genes (2;30-32). However, systemic administration of siRNA for the treatment of tumor-bearing animals had not been reported previously. Thus, the effects of shRNA on human tumor growth in vivo was next inspected using subcutaneously implanted tumor xenografts in nude mice. In these experiments recombinant U6 promoter-containing plasmids driving the expression of shRNAs (shRNA/PLK1 , shRNA/PLK1 S) in saline solution were administered to tumor-bearing mice by bolus intravenous injection every other day. For efficient delivery of plasmid DNA to organs of adult mice we injected rapidly a large volume of physiological solution into the tail vein of mice (38). According to this 'high -pressure' procedure a volume of 0.5-1.0 ml of saline containing plasmid pBS/U6/PLK1 or pBS/U6/PLK1S was injected. Since the stability of administered plasmids plays a critical role for the efficacy of shRNA expression, the stabilizing potential of ATA on plasmid DNA was tested in the blood of nude mice ex vivo. Plasmids were incubated in murine blood at 37 °C with different amounts of ATA for different times. While the supercoiled form of pure plasmid DNA was not detectable after 30 min incubation in murine blood at 37 °C, a combination with ATA extended its live-time up to 2 hr (Fig. 2). Thus, application of PBS containg plasmids and ATA was started three weeks following tumor fragment implantation, when tumors reached a volume of 50-100 mm3. In initial experiments, shRNA/PLK1 -expressing U6 plasmids were tested at a dose of 0.33-0.4 mg/kg body weight every other day and compared with the impact of shRNA/PLK1 S-expressing plasmid containing the scrambled version of shRNA/PLK1 of as a control. shRNA PLK1 displayed very potent
antiproliferative effects on the growth of HeLa S3 and A549 tumors in mice, whereas tumor growth was not inhibited by the control shRNA/PLKIS (Figs. 3, 4). During the entire experiment of 76 days mice treated with shRNA/PLK1 or shRNA/PLKIS at a dose of 0.33-0.4 mg/kg did not demonstrate decreased body weight. Interestingly, after termination of the therapy tumor growth did not progress in an accelerated fashion. From tumor -bearing mice which were treated for 28 days with shRNA/PLK1 or shRNA/PLKIS, total RNA was prepared from tumors and PLK1 levels were determined by Northern Blot analysis. Administration of shRNA/PLK1 to mice resulted in a complete suppression of PLK1 mRNA levels in tumors. In contrast, the frequency of PLK1 expression in mice treated with the scrambled control (shRNA/PLKIS) was not suppressed. This could be confirmed by Western Blot analyses showing massive protein reduction up to 90% after administration of shRNA/PLK1 compared with shRNA PLK1 S in combination with ATA at a ratio of 5:1 both.
In a second set of experiments cyclin B1 was chosen as a target for the inhibition of tumor growth in vitro and in vivo. Transfection of U6-containing plasmids for the expression of shRNA targeted to cyclin B1 led to a significant inhibition of cell proliferation in culture using HeLa S3 (cervical cancer), A549 (lung cancer), SW-480 (colon cancer) and MCF-7 (breast cancer) cells. In these cell lines cyclin B1 protein could be reduced up to 80% by shRNA targeted against cyclin B1 as confirmed by Western Blot analyses.
In addition, a human cancer xenograft experiment with HeLa S3 tumors was performed as previously described for the inhibition of PLK1 in nude mice. Systemic i.v. application of U6 vectors expressing shRNA targeted against cyclin B1 in combination with ATA at a ratio of 5:1 led to a reduction of tumor growth of 60-80% compared with the expression plasmid for the scrambled control combined with ATA at the same ratio (Fig. 5).
In summary, with the present invention we provide a powerful novel strategy
to suppress very efficiently tumor cell proliferation in cell culture. For the first time it could be demonstrated that U6 promoter-driven hairpin RNAs targeted against PLK1 or against "cyclin" B1 stabilized by the nuclease inhibitor ATA suppress tumor growth in nude mice when administered every other day systemically by intravenous injection. The power to encode a long-lasting silencing signal allows the combination of hairpin -mediated silencing with in vivo and ex vivo gene delivery strategies for therapeutic approaches based on stable RNA interference in humans. In contrast to chemotherapy, systemic RNA silencing of patients provides the fascinating perspective of taking advantage of therapeutics of physiological composition for the cure of life threatening diseases. Our findings are the launch for the systemic treatment of diseases (cancer, viral infections such as AIDS etc.) which are characterized by undesirable gene expression.
In a preferred embodiment of the present invention, the siRNA expression system contains a promoter sequence which is specific for class III RNA polymerases and especially preferrably it contains the U6 promoter or H1 promoter (18). Using the U6 promoter in an expression system leads to a very stable expression of the corresponding siRNA and it is conceivable that nuclease inhibitor concentrations can even be lowered and possibly even nuclease inhibitors are not necessary at all for obtaining sufficient siRNA expression and corresponding gene silencing when using the U6 promoter.
The basic structure of the expression system is not critical as long as it allows for efficient transcription of the genetic information of component b) of the expression system. The expression can occur either constitutively or inducably. A constitutive expression is preferred in the present context. Usually a bacterial plasmid or a viral vector will form the basis of the expression system, however, the present invention is not limited thereto.
The preconditions for formation of siRNA are known to the person skilled in the art and can also be inferred from the references mentioned supra. The
expression system contains two complementary and inverted DNA sequences which upon transcription by RNA polymerase lead to formation of double stranded RNA products. Such RNA products preferably are 15 to 30 nucleotides long and are homologous to the genes to be suppressed.
In an especially preferred embodiment of the present invention, the DNA coding for the siRNA is contained on a vector in the form of two complementary and inverted sequences which are adjacent to each other but divided by a spacer sequence, such spacer sequence being preferably 3 to 10 nucleotides long. Upon transcription of the expression system a small hairpin double stranded RNA (shRNA) is formed which interferes with expression of the gene to be silenced. It is however also possible to provide for separate transcription of the two strands of the siRNA which will anneal after transcription automatically.
In a preferred expression system on the 3'end of the sequences to be transcribed there is a RNA polymerase stop signal, preferably a T multimer.
As nuclease inhibitor every physiologically acceptable substance can be used that inhibits or decreases degradation of the expression system for a significant time. In a preferred embodiment of the present invention, as nuclease inhibitor aurin tricarboxylic acid (ATA) is used. However, the invention is not limited to this substance and every other nuclease inhibiting substance showing substantially similiar properties related to nuclease inhibiton and physiological tolerance is applicable.
It is a very favorable characterisitic of the pharmaceutical composition of the present invention that it can be applied easily, preferably by intravenous injection. In this context it is further preferred to include the pharmaceutical composition in a physiologically acceptable solution, e.g. phosphate buffered saline. Administration of larger volumes of solution promote the immediate effectiveness of gene suppression by the present pharmaceutical composition. Suitable amounts of administration of the expression system
depend on the size of the plasmid or vector used. However, a preferred dosage of expression system including the sequences being transcribed to siRNAs lies between 0.2 to 0.5 mg/kg body weight of the patient. The effect of the generated siRNA can easily be monitored by assaying for remaining expression of the gene to be suppressed. Thus the dosage can easily be adjusted to the needs. Also the addition of further substances into the injection solution is possible. Such further substances can include symptom alleviating substances, substances that strengthen the patient, antibiotics or other suitable substances. Also substances that facilitate application and carriers or auxiliaries can be included in the formulation.
In preferred embodiments of the present invention, the pharmaceutical composition is used for tumor therapy or prophylaxis. As described above and in more detail in the following examples, suppression of PLK1 or cyclin B1 expression has been shown to be an efficient method for treatment of cancer disease of several tissues. Also other proteins which are associated with tumor diseases like for example HER2/neu, BCR/ABL, ras and myc are promising candidates for gene silencing according to the present invention and therefore treatment or pevention of the corresponding cancer diseases.
In another preferred embodiment of the present invention, the pharmaceutical composition is targeted on viral genes that are actively involved in infection of cells or formation of virus progeny etc. Candidates to be combatted by the present invention are especially viruses for which no immunization is available and/or which are associated with grave diseases or damages to the patient. Examples for such viruses are Human Immunodeficiency Virus (HIV), Hepatitis viruses, Epstein Barr virus, Herpes simplex virus and Cytomegalo virus.
It is to be understood that the present invention does not only include the pharmaceutical composition described above but also the use of such composition for suppression of undesired gene expression as well as processes and methods for suppressing undesired gene expression using
such combination of expression system for specific siRNA and nuclease inhibitor. Such processes and methods are very advantageous since they are very well tolerated, are not time consuming and the pharmaceutical composition can be produced easily and inexpensively. Using the pharmaceutical composition according to the present invention or applying a corresponding method of treatment allows to inhibit tumor growth as is apparent from the examples. Using the present invention it is also possible to avoid metastasis thus leading to either prolonged survival rates and better condition of patients since chemotherapy and other physical strain can be avoided.
The present invention is further illustrated by the following examples and figures.
Fig. 1 is a schematic representation of the promoter, and sequences of the expression system which are coding for the siRNA.
Fig. 2 shows the stabilizing effect of ATA on DNA plasmids in murine blood.
Fig. 3 is a curve showing the effect of the PLK1 -targeted invention on tumor growth of HeLa S3 tumors.
Fig. 4 is a curve showing the effect of the PLK1 -targeted invention on tumor growth of A549 tumors.
Fig. 5 is a curve showing the effect of the cyclin B1 -targeted invention on tumor growth of HeLa S3 tumors.
Example 1 (PLK1)
Materials and methods
Plasmids. sequences, antibodies, and nuclease inhibitor
Plasmids were constructed using standard techniques. We used the pBS/U6 vector containing sequence elements for cloning and bacterial replication. To generate first an intermediate plasmid for the subsequent cloning steps of hairpin RNAs targeted to PLK1 , a 21 -nt oligonucleotide (5'-GGCGGCTTTGCCAAGTGCTTA-3') annealed with an 25 -nt oligonucleotide (5'-AGCTTAAGCACTTGGCAAAGCCGCC-3') corresponding to siRNA2 (23) was first inserted into the pBS/U6 vector, digested with Apal (blunted) and Hindlll. The inverted motif that contains the 6 -nt spacer and a termination string of five thymidine residues (5'-AGCTTAAGCACTTGGCAAAGCCGCCCTTTTTG-3',
5'-AATTCAAAAAGGGCGGCTTTGCCAAGTGCTTA-3') was then subcloned into the Hindlll and EcoRI sites of the intermediate plasmid to generate pBS/U6/PLK1. Sequences of the hairpin RNAs targeted to PLK1 (shRNA/PLK1 and its scrambled control shRNA2/PLK1 S) are shown in figure 1 A.
Monoclonal PLK1 antibodies for western blots were obtained from Transduction Laboratories (Heidelberg, Germany) and monoclonal antibodies against actin were obtained from Sigma -Aldrich (Taufkirchen, Germany). Goat anti-mouse secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Aurin tricarboxylic acid (ATA) was obtained from Sigma-Aldrich (Taufkirchen, Germany).
Cell Culture
Ham's F12 and fetal calf serum (FCS) were purchased from PAA Laboratories (Cδlbe, Germany). Phosphate buffered saline (PBS), Opti -MEM I, glutamine, penicillin/streptomycin, and trypsin were from Invitrogen (Karlsruhe, Germany). GenePORTER 2 was from PEQLab. The tumor cell lines HeLa S3 (cervix) and A549 (lung) were obtained from DSMZ
(Braunschweig, Germany) and cultivation was performed according to the supplier's instructions.
In vitro transfection with plasmids PBS/U6/PLK1. pBS/PLK1 S and pBS/U6 To examine the effects of the above described expression plasmids on the expression of PLK1 and on cellular proliferation in cell culture, HeLa S3 cells were transfected with plasmids using the GenePORTER 2 protocol (PEQLAB, Erlangen, Germany). One day prior to transfection cells were seeded at a density of 2x105 cells per 10 cm2. The amount of plasmid used for transfections ranged between 0.5 μg and 1.5 μg plasmid per 10 cm 2. Control cells were incubated with the scrambled control plasmids expressing shRNA/PLKI S. For PLK1 depletion cells were cotransfected with recombinant vectors and pPuro at the ratio of 10:1. 24 hours after transfection medium was changed and 2 μg/ml puromycin was added to select the transfection positive cells. Floating cells were washed away after 2 days of drug selection and the remaining cells were again incubated in the presence of puromycin.
Cells were harvested 72 or 96 hr after the beginning of transfection for Northern blot analysis, Western blot analysis, and immunofluorescence. The growth rate of 2x105 cells was determined by counting cells 48, 72, and 96 hr after the beginning of the transfection period. All transfections were performed in triplicate.
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 for the detection of PLK1 mRNA were generated by radiolabeling antisense strands for PLK1 and β-actin using 250 μCi of [α-P32]dCTP (6000 Ci/mmol; 1Ci = 37 GBq) for each reaction, 50 μM of each of the other 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 actin -2-low (5
'-CATGAGGTAGTCAGTCAGGTC-3'), as described previously (39). 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 (39). All blots were reprobed with actin probes so that actin-normalized PLK1 mRNA levels could be compared.
For the detection of shRNA/PLK1 a probe was generated by radiolabeling antisense strands for PLK1 using 250 μCi of [α-P32]dCTP (6000 Ci/mmol; 1Ci = 37 GBq) for each reaction, 50 μM of each of the other dNTPs, and 10 pmol of primer PLK1 -150-as (5'-GCAGCAGAGACTTAGGCACAA-3'), corresponding to position 310-330 within the open reading frame of PLK1 , as described previously (39). Total mRNA from transfected cells was separated on a 10% polyacrylamid-8M urea gels at 200 V in TBE for 1 hr and transferred to Hybond N+ membranes (Amersham Pharmacia Biotech., Freiburg, Germany) for 1 hr at 200 mA. The blots were prehybridi∑ed in QuickHyb® (Stratagene, Amsterdam, The Netherlands) for 20 min. and hybridized at 68 °C for 1 hr. Membranes were washed twice in 2xSSC for 5 min at 36°C and exposed to MP Hyperfilms (Amersham Pharmacia Biotech., Freiburg, Germany).
Isolation of DNA from transfected cells, tumor tissue and from murine blood Total DNA was isolated using QIAamp DNA mini -kits according to the manufacturer's protocol (Qiagen, Hilden, Germany) to determine the amount of plasmid in cell culture, tumors and the blood of nude mice (see below). DNA was separated on 1% agarose gels (100 V, 30 min) and plasmids were detected by ethidium bromide staining using an UV transilluminator.
Southern blot analysis
To determine the effect of ATA on the stability of plasmids, total DNA was isolated and electrophoresed as described above. Gels were stained with ethidium bromide and photographed. To depurinize and denature DNA, gels were incubated 15 min in 0.25 M HCI on a shaker to induce doublestrand breaks and thereafter 30 min under denaturing conditions (1.5 M NaCI and 0.5 M NaOH) on a shaker. To neutralize gels were incubated 2x 15 min in
neutralizing solution (1.5 M NaCI, 0.5 M Tris -HCI, pH 7.2, and 1 mM EDTA, pH 8.0). Then DNA was transferred onto nylon membranes as described for northern blotting analysis. Membranes were dried at room temperature and DNA was fixed on membranes for 5 min on an UV transilluminator. Blots were hybridized as described for northern blot analysis.
Western blot analysis
For western blot analysis cells were lysed and protein concentration determined as described (40). Fifty μg of total protein were separated on a 12% SDS-polyacrylamide gel and were then transferred (85V; 1.5 hr) to ImmobilonTM-P transfer membranes (Millipore, Bedford, MA). Membranes were incubated for 1 hr in 5% nonfat powdered milk in PBS with monoclonal antibodies against PLK1 (1 :250) and actin (1 :200,000) or with monoclonal (1 :100) and actin (1 :200,000) followed by incubation with goat anti -mouse serum (1 :2000) for 30 min and visualized as described before (41 ).
As was done for northern blotting experiments, PLK1 protein expression was routinely normalized to actin protein expression levels. The resulting normalized PLK1 protein levels were then presented relative to those in mock-transfected cells.
In northern and western blotting experiments PLK1 and β-actin expression was quantified using a Kodak gel documentation system (1 D 3.5). Integration of signal intensities from scanned autoradiographs was followed by quantitative comparison of PLK1 and β-actin expression, that is, for each treatment the ratio of PLK1 and β-actin signals was determined. Values are given in percentage of levels in mock-transfected cells
Determination of cell proliferation
The number of cells at each time point was determined using a hemacytometer. Cell viability was assessed by trypan blue staining. The number of mock-transfected cells (incubated with normal culture medium without GenePORTER or plasmids) after 96 hr was used as a reference. Ratios of plasmid -treated cells and mock-transfected cells was determined to
obtain the percentage of proliferating cells. Each experiment was performed in triplicate. Means and 95% confidence intervals were determined.
Fluorescence-activated cell sorting analysis Cell cycle distribution and apoptosis were analyzed using a Becton Dickinson FACScan apparatus. Cells were harvested, washed with PBS, and probed with CycleTESTTM PLUS DNA reagent kit (Becton Dickinson, Heidelberg, Germany) according to the manufacturer's protocol to determine cell cycle distribution. For each transfection (mock -transfected and each plasmid), 30,000 cells were analyzed in triplicate. Percentage of cells in different cell cycle phases was calculated using ModFit LT for Mac. For the detection of apoptotic phenotypes, harvested cells were fixed with ice -cold 70% ethanol, treated for 20 min at 37 C with RNase A (5 μg/mL) and with propidium iodide (50 μg/mL). Subsequent analyses of cell cycle distribution and apoptosis were performed using the CELLQuest software (Becton Dickinson, Heidelberg, Germany).
DNA staining of transfected cells
48 hr after the beginning of transfection cells were washed twice in PBS and fixed for 15 min in icecold methanol. After washing three times in PBS for 10 min DNA was stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Sigma-Aldrich, Taufkirchen, Germany) for 20 min. Cells were examined with a fluorescence microscope (Leica, Wetzlar, Germany) at a magnification of 40x.
Measurement of U6-based shRNA efficacy in nude mouse tumor models Human cancer xenograft models were established with at least 3 independent groups of 5 athymic nude mice (nu/nu) NMRI 8-10 weeks old. For this purpose HeLa S3 cells were harvested, washed with PBS, resuspended in normal culture media. Thereafter 2x106 cells were injected subcutanously into the animals flank regions. Arising tumors were serially passaged by a minimum of three consecutive transplantations prior to treatment. Then tumor fragments were implanted subcutaneously in both
flanks of the nude mice and treatment with plasmids was started when the tumor reached a volume of 100 mm3. Treatment was carried out every other day by injection of 500 μl PBS containing 10 μg plasmid and 2 μg ATA (ATA:DNA at a ratio of 1 :5) into the tail vein. One group was treated with plasmid expressing shRNA/PLK1 without ATA, the second group with a combination of pBS/shRNA PLK1 and ATA. Another group was treated with pBS/shRNA/PLK1S with or without ATA as control. The mock-treated group received 500 μl PBS every other day. Tumor diameters were determined using a caliper. Volumes were calculated according to the formula V= /6 x largest diameter x smallest diameter2. Experiments were carried out in triplicate, means and 95% CI were calculated. After sacrificing the animals tumors were excised for detection of shRNA/PLK1 using northern blot analysis.
Statistical methods
Each western blot and northern blot experiment was performed three times. Means of normalized (i.e., to actin) signal intensities were calculated. For the determination of proliferation, cell numbers were determined in triplicate at each time point. FACScan analyses were carried out three times for each cell type. Statistical analysis was performed with two-way ANOVA (GraphPad Prism, GraphPad Software, Inc., San Diego, California) to consider random effects of individual gels and different treatments. For two-way ANOVAs all treatment groups were compared to mock-transfected cells. P values and 95% confidence intervals (CI) for the statistical significance of the changes caused by each transfection are given.
Example 2 (Cyclin B1)
Materials and methods
Plasmids. sequences, antibodies, and nuclease inhibitor
Plasmids were constructed using standard techniques. We used the pBS/U6
vector containing sequence elements for cloning and bacterial replication. To generate first an intermediate plasmid for the subsequent cloning steps of hairpin RNAs targeted to cyclin B1 , a 19-nt oligonucleotide (5'-GTCAGTGAACAACTGCAGG-3') annealed with a corresponding antisense oligonucleotide was first inserted into the pBS/U6 vector. The inverted motif that contains the 6-nt spacer and a termination string of five thymidine residues was then subcloned into the Hindlll and EcoRI sites of the intermediate plasmid to generate pBS/U6/PLK1. Sequences of the hairpin RNAs targeted to cyclinBI (shRNA/cyclinB1 and its scrambled control shRNA/cyclinB1S).
Monoclonal Cyclin B1 antibodies for western blots were obtained from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany) and monoclonal antibodies against actin were obtained from Sigma-Aldrich (Taufkirchen, Germany). Goat anti -mouse secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Aurin tricarboxylic acid (ATA) was obtained from Sigma-Aldrich (Taufkirchen, Germany).
Cell Culture Ham's F12 and fetal calf serum (FCS) were purchased from PAA Laboratories (Cδlbe, Germany). Phosphate buffered saline (PBS), Opti -MEM I, glutamine, penicillin/streptomycin, and trypsin were from Invitrogen (Karlsruhe, Germany). GenePORTER 2 was from PEQLab. The tumor cell lines HeLa S3 (cervix), A549 (lung), MCF-7 (breast) and SW-480 (colon) were obtained from DSMZ (Braunschweig, Germany) and cultivation was performed according to the supplier's instructions.
In vitro transfection with plasmids pBS/U6/cyclinB1 , pBS/cyclinB1S and pBS/U6 To examine the effects of the above described expression plasmids on the expression of PLK1 and on cellular proliferation in cell culture, HeLa S3 cells were transfected with plasmids using the GenePORTER 2 protocol (PEQLAB, Erlangen, Germany). One day prior to transfection cells were
seeded at a density of 2x105 cells per 10 cm2. The amount of plasmid used for transfections ranged between 0.5 μg and 1.5 μg plasmid per 10 cm 2. Control cells were incubated with the scrambled control plasmids expressing shRNA/cyclinB1S. For Cyclin B1 depletion cells were cotransfected with recombinant vectors and pPuro at the ratio of 10:1. 24 hours after transfection medium was changed and 2 μg/ml puromycin was added to select the transfection positive cells. Floating cells were washed away after 2 days of drug selection and the remaining cells were again incubated in the presence of puromycin. Cells were harvested 72 or 96 hr after the beginning of transfection for Northern blot analysis, Western blot analysis, and immunofluorescence. The growth rate of 2x105 cells was determined by counting cells 48, 72, and 96 hr after the beginning of the transfection period. All transfections were performed in triplicate.
Western blot analysis
For western blot analysis cells were lysed and protein concentration determined as described (40). Fifty μg of total protein were separated on a 12% SDS-polyacrylamide gel and were then transferred (85V; 1.5 hr) to ImmobilonTM-P transfer membranes (Millipore, Bedford, MA). Membranes were incubated for 1 hr in 5% nonfat powdered milk in PBS with monoclonal antibodies against Cyclin B1 (1 :250) and actin (1 :200,000) or with monoclonal (1 :100) and actin (1 :200,000) followed by incubation with goat anti-mouse serum (1 :2000) for 30 min and visualized as described before (41).
Cyclin B1 protein expression was routinely normalized to actin protein expression levels. The resulting normalized Cyclin B1 protein levels were then presented relative to those in mock-transfected cells.
In western blotting experiments Cyclin B1 and actin expression was quantified using a Kodak gel documentation system (1 D 3.5). Integration of signal intensities from scanned autoradiographs was followed by quantitative
comparison of Cyclin B1 and actin expression, that is, for each treatment the ratio of Cyclin B1 and actin signals was determined. Values are given in percentage of levels in mock-transfected cells
Measurement of U6-based shRNA efficacy in nude mouse tumor models Human cancer xenograft models were established with at least 3 independent groups of 5 athymic nude mice (nu/nu) NMRI 8-10 weeks old. For this purpose HeLa S3 cells were harvested, washed with PBS, resuspended in normal culture media. Thereafter 2x106 cells were injected subcutanously into the animals flank regions. Arising tumors were serially passaged by a minimum of three consecutive transplantations prior to treatment. Then tumor fragments were implanted subcutaneously in both flanks of the nude mice and treatment with plasmids was started when the tumor reached a volume of 100 mm3. Treatment was carried out every other day by injection of 500 μl PBS containing 10 μg plasmid and 2 μg ATA (ATA:DNA at a ratio of 1 :5) into the tail vein. One group was treated with plasmid expressing shRNA/cyclinB1 without ATA, the second group with a combination of pBS/shRNA/cyclinB1 and ATA. Another group was treated with pBS/shRNAcyclinB1S with or without ATA as control. The mock -treated group received 500 μl PBS every other day. Tumor diameters were determined using a caliper. Volumes were calculated according to the formula V=π/6 x largest diameter x smallest diameter2. Experiments were carried out in triplicate, means and 95% CI were calculated.
Statistical methods
Each western blot experiment was performed three times. Means of normalized (i.e., to actin) signal intensities were calculated. For the determination of proliferation, cell numbers were determined in triplicate at each time point. Statistical analysis was performed with two-way ANOVA (GraphPad Prism, GraphPad Software, Inc., San Diego, California) to consider random effects of individual gels and different treatments. For
two-way ANOVAs all treatment groups were compared to mock-transfected cells. P values and 95% confidence intervals (CI) for the statistical significance of the changes caused by each transfection are given.
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