Down-regulation of MDM2 and activation of p53 in human cancer cells by
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《核酸研究医学期刊》
Department of Medical Biochemistry and Genetics, The Panum Institute, University of Copenhagen, Blegdamsvej 3c, 2200 Copenhagen N, Denmark
* To whom correspondence should be addressed. Tel: +45 35327762; Fax: +45 35396042; Email: pen@imbg.ku.dk
ABSTRACT
A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these PNAs on the cells was also reflected in reduced levels of the MDM2 protein and increased levels of the p53 tumor suppressor protein, which is negatively regulated by MDM2. Initially, PNA oligomers were delivered as DNA complexes with lipofectamine, but it was discovered that PNA conjugated to the DNA intercalator 9-aminoacridine (Acr) (Acr–PNA) could be effectively delivered to JAR cells (as well as to HeLa pLuc705 cells) even in the absence of a DNA carrier. Using such lipofectamine-delivered Acr–PNA conjugates, one PNA targeting a cryptic AUG initiation site was identified that at a concentration of 2 μM caused a reduction of MDM2 levels to 20% (but no reduction in mdm2 mRNA levels) and a 3-fold increase in p53 levels, whereas a 2-base mismatch control had no such effects. Furthermore, transcriptional activation by p53 was also increased (6-fold), and cell viability was reduced to 80%. Finally, this PNA acted cooperatively with camptothecin treatment both with regard to p53 activity induction as well as cell viability. Using this novel cell delivery system, we have identified a target on the mdm2 mRNA that appears sensitive to antisense inhibition by PNA and therefore could be used as a lead for further development of mdm2-targeted antisense (PNA and other) gene therapeutic anticancer drugs.
INTRODUCTION
The potential of peptide nucleic acid (PNA) in anticancer drug discovery is still rather poorly explored. This is to a large extent due to the lack of a simple and robust method for the delivery of PNA to eukaryotic cells. Nonetheless, encouraging results have been reported in a range of systems (1–5). A number of studies have exploited cell-penetrating peptides as carriers of PNA (1,6–8), but (at least some of) these predominantly deliver PNA to the endosomal (lysosomal) compartment of the cell and not very efficiently to the cytosolic or nuclear compartment where the agent should exert its antisense action. Furthermore, most PNA–peptide conjugates significantly complicate the synthetic availability of these agents. Antisense oligonucleotides are effectively delivered to eukaryotic cells via cationic liposome complexation (9). Although PNA oligomers being molecules with a neutral charge cannot directly complex with (cationic) liposomes, delivery via this route is possible using PNA–lipid conjugates (10,11) or PNA–DNA hybrid complexes (12,13). In particular, the latter method appears general and effective, although the need for specifically optimized DNA oligonucleotides for each new PNA oligomer is a cumbersome drawback. Nevertheless, we decided to exploit this system in an anticancer drug discovery approach using the oncogene mdm2 as target.
The MDM2 protein is over-expressed and/or up-regulated in 40–60% of human sarcomas as well as in several solid tumors which may have a wild-type p53 (14–17). The regulation of the expression of MDM2 and p53 is tightly coupled as the expression of MDM2 is induced by p53 (18,19). Furthermore, MDM2 binds to p53 with a high affinity, inhibiting its ability to act as a transcription factor and also induces its degradation (20,21), indicating that MDM2 functions as a negative feed back regulator of p53 (21). Therefore, MDM2 is recognized as an important target for cancer therapy (22,23), resulting in up-regulation of p53 (20,24,25). In agreement with this hypothesis, an mdm2 antisense phosphorothioate oligonucleotide (PS) was shown to reduce MDM2 expression and to release p53 from a p53–MDM2 complex in a variety of cancer cell lines (20,24–26).
Furthermore, the mdm2 gene apart from being a relevant anticancer drug target, offers the possibility of effect analysis on both MDM2 down-regulation and p53 up-regulation. Finally, synergy on cell survival and p53 activity would be expected with traditional DNA damaging (e.g. via topoisomerase interference) anticancer drugs.
In contrast to (phosphorothioate) oligonucleotides, PNA oligomers do not activate RNaseH upon binding to mRNA. Consequently, sequence targets on a given mRNA that are sensitive to RNaseH-recruiting antisense agents are not necessarily good targets for PNA antisense agents. Thus the objectives of this study were to identify mdm2 mRNA sequence targets that are sensitive to PNA antisense agents. As a parallel objective we also seek to discover novel delivery methods for PNA oligomers.
MATERIALS AND METHODS
PNA synthesis
The sequences of the PNAs targeting mdm2 mRNA are shown in Figure 1. For the splicing correction study with HeLa pLuc705 cells, two 9-aminoacridine (Acr)–PNAs were used. PNA synthesis was carried out as reported previously (27). High-performance liquid chromatography (HPLC) analysis was performed on Delta Pak C18 column (5 μm, 3.9 x 150 mm) at room temperature with a flow rate of 1 ml/min using an acetonitrile/water (containing 0.1% TFA) gradient. The purification was performed on a C18 (Delta Pak, Waters, Copenhagen, Denmark) (15 μm, 19 x 300 mm) with a flow rate of 8 ml/min. All compounds were characterized by mass spectrometry using a Kratos MALDI-II time-of flight mass spectrometer and 3,5-dimethoxy-4-hydroxycinnamic acid as matrix. The PNAs were lyophilized and stored at 4°C. SV40 core nuclear localization signal (NLS) (PKKKRKV) was covalently linked to PNA at the N-terminal through an ethyleneglycol linker (eg1, 8-amino-2,6-dioxaoctanoic acid) via continuous synthesis. N-(9-aminoaciridinyl)-6-aminohexanoic acid (Acr) was linked to PNA on the solid support at the N-terminal of the PNA (28) through the eg1 linker (Acr–PNA). Fluorescein (Fl)-labeled PNA oligomers were prepared using a previously described ‘Fl–lysine’ monomer (29).
Figure 1. Sequences and location of the PNAs within the sequence of MDM2 mRNA (accession no. NM_002392 , 272–512 bp). (A) Target positions of PNAs are indicated as T1, T2, T3, T4 and T5. Arrows indicate the location and length of the antisense PNAs. (B) T5-12-mm and T5-12-mm–Acr are the mismatch PNA of T5-12 and T5-12–Acr respectively. eg1, 8-amino-2,6-dioxaoctanoic acid; Fl, fluorescein. PNA No.(1) indicates nomenclatures of the PNAs used in this study. PNA No.(2) indicates the reference PNA numbers. Unmodified PNAs are indicated in plain text. 9-aminoacridine (Acr)-conjugated PNAs (Acr–PNAs) are indicated with underlines. NLS-PNAs, PNAs conjugated with the SV40 core NLS (PKKKRKV), are indicated in italic.
Cell cultures
JAR cells were a kind gift from Dr Peter Ebbesen (The Stem Cell Laboratory, Aalborg University, Denmark). HeLa pLuc705 cells (29) were purchased from Gene Tools (USA). Both cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% GLUTAMAX I (GIBCO, Invitrogen). All cell cultures were incubated at 37°C with 5% CO2 for a minimum of 16 h before initiating transfection.
PNA transfection
Cells were plated and incubated at 37°C to give 40–60% confluence at the time of transfection. Before the transfection, the culture medium was replaced with OPTI-MEM (1% FBS) (Life Technologies) of sufficient volume to cover the cells. For the transfection of PNAs by the complementary DNA and LipofectAMINE (LFA, Life Technologies), the method of Doyle et al. (13) was used. Oligo-DNAs of the same length and complementary sequence to the corresponding PNAs were used in this study.
For Acr–PNA transfection, Acr–PNA/calf thymus DNA (CT-DNA)/LFA complex was prepared in OPTI-MEM. Five microliters of Acr–PNA (200 μM for a final concentration of 2 μM in the medium) were mixed with 40 μl of OPTI-MEM and incubated for 35 min at room temperature. The solution was mixed with 50 μl of diluted LFA solution (diluted with OPTI-MEM to 60 and 80 μg/ml respectively for JAR cells and HeLa pLuc705 cells) and incubated for 35 min at room temperature unless otherwise stated. This solution was added to the cells in 0.4 ml of OPTI-MEM (1% FBS) and incubated under the normal cell growth condition.
Cell proliferation assay
Cells were plated in 96-well microtiter plates (Nunc) and grown to 40% cell confluence at the time of transfection. PNA/complementary-DNA/LFA complex was transfected at the indicated concentrations as mentioned above. The camptothecin (CPT) solution (when desired) was added to the cultured cells at the indicated concentration simultaneously with the transfection of Acr–PNA/LFA complex as mentioned above. Cell viabilities were assayed using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Bie&Berntsen A/S, Denmark) according to the manufacturer's instructions.
Western blot analysis
Cells were lysed in boiling lysis buffer (1% SDS, 10 mM Tris–HCl, pH 7.2, 1x protease inhibitor cocktail tablet (Roche, Germany)], and 40 μg of the protein lysate was fractionated by SDS–PAGE and transferred to nitrocellulose membranes (ADVANTEC MFS). The nitrocellulose membrane was incubated with blocking buffer (2% BSA, 20 mM Tris–HCl, pH 7.2, 150 mM NaCl and 0.01% Tween-20) overnight at 4°C. The membrane was incubated with primary antibody overnight at 4°C. The membrane was washed with washing buffer (20 mM Tris–HCl, pH 7.2, 150 mM NaCl and 0.01% Tween-20) three times for a minimum of 15 min each at room temperature, and then incubated with 1:20 000 diluted anti-mouse IgG–alkaline phosphate-conjugated secondary antibody (Sigma) for 1 h at room temperature. After washing as described above, the protein of interest was detected with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Fluorescence microscopy
For uptake analysis, exponentially growing cells were plated in 4-well chamber slides (Nunc) the day before transfection. Transfections were performed as described above using Fl-labeled PNAs (PNA2297, 2415) with the LFA (6 and 8 μg/ml for JAR cells and HeLa pLuc705 cells, respectively). After 16 h, the cells were rinsed twice with growth medium (RPMI 1640 containing 10% FBS and 1.5% glutamax) and subjected to microscopic studies without fixation. For the propidium iodide counter staining, cells were incubated with 8 μg/ml of propidium iodide for 15 min at 37°C before being subjected to the microscopic study. Cells were visualized using a LEITZ WETZLAR (Germany) fluorescent microscope (DIALUX 22EB) at 400 x magnification with filter block N2 (Ernst Leitz Wetzlar GmbH, Germany).
Luciferase assay
Exponentially growing HeLa pLuc705 cells were plated in 24-well plates the day before transfection. For the transfection, Acr–PNA (PNA2391), targeting the aberrant splice site within the luciferase pre-mRNA was used in combination with LFA (8 μg/ml) and CT-DNA as described above (see the PNA synthesis for the sequences of PNA2391 and PNA2746). After 24 h, cells were lysed with Passive Lysis Buffer (Promega) and subjected to luciferase activity measurements. Luciferase activities were measured with Luminoskan TL Plus (Thermo Labsystems) by using the Luciferase Assay System (Promega) following the manufacturer's procedures. The relative light units (RLU) were normalized to micrograms of protein determined by the Bradford method (Coomassie Plus, Pierce) following the manufacturer's procedure.
RT–PCR
Exponentially growing HeLa pLuc705 cells were treated with Acr–PNA/LFA as described in the luciferase assay section. Total RNA was extracted from the cells by using RNeasy Mini kit (Qiagen) and subjected to RT–PCR. RT–PCR was performed by using OneStep RT–PCR kit (Qiagen). Three nanograms of total RNA was used for each RT–PCR reaction (20 μl). Primers for RT–PCR were as follows: forward primer, 5'-TTGATATGTGGATTTCGAGTCGTC-3'; reverse primer, 5'-TGTCAATCAGAGTGCTTTTGGCG-3'. RT–PCR program was as follows: 55°C for 35 min, 1 cycle; 95°C for 15 min, 1 cycle; 94°C for 0.5 min, 55°C for 0.5 min, 72°C for 0.5 min, 30 cycles]. RT–PCR products were analyzed on a 2% agarose gel run in 1x TBE buffer and visualized by ethidium bromide staining. Gel images were captured by ImageMaster (Pharmacia Biotech) and analyzed by UN-SCAN-IT software (Silk Scientific Corporation).
Northern analysis
For northern blot analysis, 15 μg of total RNA was analyzed using AlkPhos DIRECT (Amersham Pharmacia Biotech) according to the manufacturer's instructions. A double-stranded DNA fragment of MDM2, bases 312–1787 (GenBank accession no. NM_002392 ), were used as a gene specific probe.
DNA damage treatment and luciferase assay
A firefly luciferase vector holding the p53-responsive element of human MDM2 upstream of the firefly luciferase gene, was used as a reporter vector (pGL3-53RE). The pRL-CMV vector (Promega), holding a Renilla luciferase, was used as a control vector. These two plasmid DNAs were mixed (pGL3-53RE:pRL-CMV of 49:1) and used for transfection. Transcriptional activation by p53 was measured as the ratio of firefly luciferase activity (reporter)/Renilla luciferase activity (control). For construction of the reporter vector pGL3-53RE, p53-responsive element sequence (GenBank/EMBL accession no. AF144015 , nucleotides 95–179) from human MDM2 gene was cloned into the pGL3-Basic Vector (Promega) upstream of the luc+ gene (18).
For the DNA damage treatment, CPT was added to the cultured cells at the indicated concentration simultaneously with the addition of Acr–PNA/LFA mixture as mentioned above. The luciferase activities were determined by Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
RESULTS AND DISCUSSION
Initially, we designed a series of PNA oligomers targeting the translation initiation site and regions immediately downstream from this on the mdm2 mRNA. We also included an internal ATG site . All of these PNAs were also tested as conjugates with the NLS peptide (PKKKRKV) as this has been reported to enhance cellular delivery (1,28), and at least should ensure nuclear localization of the PNA. All PNAs were delivered to the JAR cells at 1 μM concentration using the oligonucleotide–LFA protocol described by Doyle et al. (13).
A cell viability screening revealed (Figure 2) that only three of the PNAs (T1-12, T5-12 and T5-12-NLS) showed cellular cytotoxicity as an indication of antisense activity. We therefore decided to examine these PNAs (as well as the NLS version of T1-12:T1-12-NLS) further in terms of the effects on MDM2 and p53 expression. The results fully support the contention that the effects on cell viability are indeed caused by antisense down-regulation of MDM2 and a concomitant up-regulation of p53 (Figure 3A) since PNAs T1-12, T5-12 and T5-12-NLS show clear effects on protein levels. It is noteworthy that conjugation of NLS to the PNA did not significantly enhance the antisense effect. Indeed, for the T1 target, conjugation to NLS decreased the effect. Furthermore, T5-12-NLS showed no effects on cell viability up to 10 μM when delivered to the cells without the carrier oligonucleotide and LFA (data not shown) indicating that the NLS peptide is not capable of delivering this PNA oligomer to JAR cells. This is in contrast to results reported in Burkitt's lymphomas (BL) cells (1).
Figure 2. Effect of target site on the cellular viabilities by antisense PNAs. PNAs (final concentration of 1 μM) were transfected to JAR cells as complexes with complementary DNA and LFA (6 μg/ml) (2). After 18 h of transfection, cell viabilities were determined by MTS assay (Promega).
Figure 3. Effect of antisense-PNAs on MDM2 and p53 levels in JAR cells. Cells were transfected with PNA as complexes with complementary DNA and LFA for 24 h. Identical amounts of protein were analyzed by western blot using monoclonal anti MDM antibody and anti p53 antibody. (A) T1-12, T1-12-NLS, T5-12 and T5-12-NLS were used at 1 μM. (B) T5-12 was tested at 0.5, 1 and 2 μM. Mismatch PNA (T5-12-mm), holding 2 bp mismatches, was used at 2 μM.
In order to further substantiate an antisense mechanism, we tested a double mismatch (base interchange) derivative of the active T5-12 (T5-12-mm) and this showed no effect on either the MDM2 or the p53 levels at 2 μM, at which concentration T5-12 reduced the MDM2 level to 15% and resulted in a 2.4-fold increase in p53 level (Figure 3B). Finally, we compared the dose response of T5-12 and T5-12-mm cell survival assay (Figure 4A), in which T5-12 showed cytotoxicity <1 μM, whereas T5-12-mm did not affect cell viability up to 5 μM.
Figure 4. Effect of the T5-12 on the cellular viabilities and p53-activation. (A) JAR cells were transfected with T5-12 and T5-12-mm (mismatch control) at the indicated concentrations for 36 h as complexes with complementary DNA and LFA. Cellular viabilities were determined by MTS assay (Promega). Data are mean ± SD of six independent experiments. The value without PNA was set as 100% survival. (B) Transcriptional activation of p53 induced by T5-12 in JAR cells. Cells were transfected with the PNA/complementary-DNA/LFA complex (1 μM PNA) for 3 h followed by a transfection of vectors (p53-inducible firefly luciferase vector (pGL3-53RE) and pRL-CMV vector as an internal control) as a complex with LFA (6 μg/ml) for 24 h. T5-12-mm was used as a 2 bp mismatch control. Transcriptional activation of p53 is shown as a ratio of firefly luciferase activity/Renilla luciferase activity. Data are mean ± SD of three independent experiments.
In order to study whether the increased level of p53 was also translated into increased p53 activity, we employed a dual luciferase reporter assay in which one vector harbors the firefly luciferase gene controlled by the p53 responsive element at the promoter and the Renilla luciferase controlled by the constitutive cytomegalovirus (CMV) promoter is used as internal control. Thus the ratio of firefly versus Renilla luciferase activity will be a measure of p53 activity. As shown by the results presented in Figure 4B, treatment of the cells with PNA T5-12 does indeed result in a relative increase in firefly luciferase activity as compared with non-treated controls cells or to cells treated with the double mismatched T5-12-mm, which in this assay showed some activation of p53, as would be expected from the p53 levels (Figure 3B).
Therefore, using the oligonucleotide–LFA delivery method, we have identified a possible PNA anticancer drug antisense lead target (that of T5-12) on the mdm2 mRNA. However, this delivery method is not adequate for screening of large numbers of PNA oligomers. Using PNA T5-12 as the lead compound, we decided to explore an alternative delivery method. We reasoned that a PNA oligomer conjugated to the DNA intercalator Acr should confer general affinity for double-stranded DNA, and that using such acridine–PNA conjugates could allow the use of, e.g., CT-DNA as a general carrier in combination with cationic lipids. Consequently, we synthesized a T5-12–Acr conjugate (PNA2296) and a similar compound also containing Fl (T5-12–Acr–Fl, PNA2297) (Figure 5A).
Figure 5. Structures and transfection studies of Acr-conjugated PNAs. (A) Chemical structures of 9-aminocaprorylacidine (Acr)-conjugated PNA , Fl-labeled Acr–PNA and Fl-labeled PNA . (B) Transfection studies of Acr–PNA with JAR cells and HeLa pLuc705 cells. T5-12–Acr–Fl (1 μM) (Acr–Fl–PNA, Fl-labeled Acr–PNA) or T5-12–Fl (1 μM) (Fl–PNA, Fl-labeled PNA without Acr) was transfected into the cells for 16 h in combinations with or without CT-DNA (1 μg/ml) and/or LFA (6 and 8 μg/ml for JAR cells and HeLa pLuc705 cells, respectively). Cells were incubated with Acr–Fl–PNA/CT-DNA/LFA (a), Acr–Fl–PNA (b), Acr–Fl–PNA/CT-DNA (c), Acr–Fl–PNA/LFA (d) or Fl–PNA/LFA (e). (C) Acr–Fl–PNA transfection study of JAR cells and HeLa pLuc705 cells with propidium iodide counter staining. Transfection of the Acr–Fl–PNA were performed under the same condition in (B)–(d). Cells were treated with 8 μg/ml of propidium iodide for 15 min before being subjected to microscopic study.
Cellular uptake of Acr–PNA
Transfection of Fl-labeled Acr-conjugated PNAs (Acr–Fl–PNA, T5-12–Acr–Fl) were tested in combination with CT-DNA and LFA (Figure 5B). Transfection with the Acr–Fl–PNA/CT-DNA/LFA complex resulted in a high level of fluorescence in treated JAR cells and HeLa pLuc705 cells . As a control experiment, Acr–Fl–PNA was added to the cells either free or in complex with CT-DNA and/or LFA. Unexpectedly, the transfection of the cells by Acr–Fl–PNA in combination with LFA also resulted in a high level of fluorescence in the treated cells without showing severe cellular toxicity (counter staining with propidium iodide in Figure 5C). However, treatment with Acr–Fl–PNA alone or Acr–Fl–PNA in combination with CT-DNA did not result in any significant intracellular fluorescence . In addition, Fl-labeled PNA (Fl–PNA, T5-12–Fl), not containing the Acr, was not effectively delivered by LFA either to JAR cells or to HeLa pLuc705 cells . Thus, inclusion of DNA was not required for effective cellular uptake. Although the fluorescence microscopy data do not allow a detailed determination of the intracellular localization of the Acr–PNA, most of the PNA does appear to be present in vesicles (endosomes?) and not evenly distributed in the cytoplasm. No preferential staining of the nucleus is observed either. Furthermore, a counterstain with propidium iodide which stains only dead cells clearly indicates that cells that take up the PNA are still alive and healthy. We believe that the aminoacridine on the non-protonated form in analogy to fatty acids (10) is sufficiently lipophilic to allow complexation with the liposomes, whereas the protonated form increases solubility in the aqueous environment. (If this is indeed the mechanism, it should be possible to further develop this concept using other analogous polycyclic ligands.)
To support the conclusion of effective cellular internalization of the Acr–PNA by LFA delivery, we employed the sensitive luciferase antisense activation method with HeLa pLuc705 cells developed by Kole et al. (30). HeLa pLuc705 cells are stably transfected HeLa cells with a modified luciferase containing the mutated intron2 of the human ?-globin pre-mRNA causing pre-mRNA missplicing and resulting in a non-functional enzyme. By using PNA targeting the aberrant splice site, this assay can measure antisense activity (reflecting effective uptake) of PNA as restored luciferase activity as a consequence of mRNA splice correction by the PNA (30).
We chose a PNA sequence previously identified by Kole et al. (30,31). As anticipated the corresponding Acr–PNA (PNA2391) showed good antisense activity (as luciferase activity) when complexed with LFA or LFA/CT-DNA but showed no significant activity without LFA (Figure 6A). These data fully corroborate the uptake studies (Figure 5B). In addition, the cells transfected with Acr–PNA/LFA showed comparable antisense activity as compared with the conventional method, using complexes of PNA/complementary-DNA/LFA (data not shown). However, a more systematic comparative study in more systems (cell types and gene targets) are required in order to draw more general quantitative conclusions of the relative efficacy of the two delivery methods. The antisense effect of the Acr–PNA was also demonstrated by a dose-dependent correction of missplicing (Figure 6B and C) (47% at 4 μM of Acr–PNA), which correlated fully with the increase of luciferase activity. Finally, a control mismatch PNA (MM, PNA2746) did not show (antisense) activity either in the presence or in the absence of LFA. These results demonstrate that the Acr–PNAs, transfected as a complex with LFA, are to a significant extent delivered into the nucleus where the Acr–PNA binds the target mRNA and corrects splicing.
Figure 6. Acr–PNA transfection studies with the HeLa pLuc705 cells. HeLa pLuc705 cells were transfected with Acr–PNA (PNA 2391), targeting the cryptic splice site of luciferase pre-mRNA to correct the missplicing, or its mismatch PNA (MM, PNA2746) at the indicated concentrations in combination with or without CT-DNA (1 μg/ml) and/or LFA (8 μg/ml). After 24 h, cells were subjected to the further analysis. Luciferase activities were measured by Luciferase Assay System (Promega). The obtained RLU were normalized to micrograms of protein. Each data point is an average of three independent experiments ± SD. (A) Acr–PNAs (1 μM) with/without addition of LFA and/or CT-DNA were transfected for 24 h. (B) The cells were treated with PNA2391 (Acr–PNA) or mismatch PNA (MM, PNA2746) in the absence (–LFA) or presence of LFA (+LFA) for 24 h. Total RNA was extracted from the cells and subjected to RT–PCR analysis. U, uncorrected form (268 bp), which contains aberrant exon. C, corrected form (142 bp) in which the entire intron was eliminated. The values under the lower panel (+LFA) indicate the relative amount of corrected form. The corrected form in LFA-free transfections amounted to <1% in all cases. (C) HeLa pLuc705 cells were transfected as in (B) and the cells were subjected to a luciferase activity analysis.
Thus the LFA/Acr–PNA delivery method is a convenient method for testing larger series of PNA oligomers, as no (sequence designed) carrier oligonucleotide is required. Consequently, we used Acr–PNA/LFA transfection for further experiments.
Inhibition of MDM2 expression by antisense Acr–PNAs
A series Acr–PNAs targeted within the mdm2 mRNA were tested at a concentration of 1 μM for their ability to down-regulate MDM2 (Figure 7). Among the six Acr–PNAs (12 bp of each) tested, T2-12–Acr and T5-12–Acr showed significant down-regulation of the MDM2 protein level to 33 and 27% of the control, respectively (Figure 7A).
Figure 7. Inhibition of MDM2 protein expression by antisense Acr–PNAs. JAR cells were treated by PNAs with LFA (6 μg/ml) for 24 h. Identical amounts of protein were analyzed by western blot using a monoclonal anti-MDM antibody and/or monoclonal anti-p53 antibody. (A) Six Acr–PNAs (1 μM) of 12 bp targeted to the different position within the mdm2 mRNA sequence were tested for down-regulation of MDM2 expression. (B) The effect of Acr–PNA length on the antisense effects. Acr–PNAs (1 μM) with different length (8, 10, 12, 14, 16 and 18 bp) were tested for down-regulation of MDM2 expressions. (C) The effect of Acr–PNA length on the MDM2 and p53 expression levels in JAR cells. Seven Acr–PNAs (1 μM) with different lengths (12, 13, 14, 15, 16, 18 and 20 bp) were tested. (D) The effect of T5-12–Acr on the MDM2 and p53 expression in JAR cells. T5-12–Acr (Acr–PNA), T5-12-mm–Acr (mismatch control for T5-12–Acr) and T5-12 (naked PNA) were used at the concentrations indicated.
Using these two PNAs as leads, a series of Acr–PNAs of different length from 8 bp to 18 bp were also tested (Figure 7B and C). These results revealed a clear optimum length of 12–16 bases for the T2-12–Acr series and an optimum around 13 bases for the T5-12–Acr series assayed both for MDM2 reduction as well as p53 increase (Figure 7C). In contrast to the previous report by Corey's group (32) about the requirements of the longer PNA (>17 bp) for the antisense effects, the longer PNAs (18–20 bases) showed a weaker effect than the shorter PNAs. However, it should be emphasized that in these experiments we were not able to separate the differences in the efficiency of delivery and antisense activity, as the results reflect the effect of the treatment as such, and the weaker effect of the longer PNAs could be due to a less effective delivery. This phenomenon was previously observed in bacteria (33).
It is noteworthy, that the most active of the Acr–PNAs, T5-12–Acr, is an acridine conjugate of the most active unmodified PNA (T5-12), thereby supporting the sensitivity of this sequence target on the mdm2 mRNA. Thus, we decided to study this PNA further. First, we wished to obtain evidence that the observed MDM2 down-regulation was accomplished through a sequence-specific antisense mechanism by using a mismatch PNA (T5-12-mm–Acr) as a control (Figure 7D). This experiment showed that while fully matched PNA (T5-12–Acr) down-regulates MDM2 in a dose-dependent manner, mismatched PNA (T5-12-mm–Acr) containing two mismatches did not significantly inhibit MDM2 expression even at a concentration of 2 μM. As a further control of the delivery system T5-12 lacking the Acr did not show significant down-regulation of MDM2 under these conditions. As expected the p53 levels showed opposite tendencies as compared with the MDM2 protein levels. T5-12–Acr caused a 2.9-fold up-regulation of p53 at 2 μM, while none of the control PNAs (T5-12-mm–Acr and T5-12) had such effects. Finally, the effect on mdm2 mRNA levels was assayed by northern blot analysis, and no detectable change upon treatment with T5-12–Acr was observed (Figure 8). Thus the mechanism appears to be translation inhibition rather than mRNA destabilization. This is somewhat surprising in view of the position of the target 150 bases downstream of the translation initiation, which is usually considered more sensitive for non-RnaseH-supported antisense inhibition. The target is at a cryptic AUG initiation site, but the function of this is obscure. Thus the molecular mechanism by which T5-12–Acr and T5-12 (and analogues) inhibit MDM2 translation is not clear at this stage, but most likely involves translation elongation arrest, as previously observed in some cases (34).
Figure 8. Northern blot analysis of mdm2 mRNA expression in JAR cells treated with Acr–PNAs (2 μM) with LipofectAMINE (LFA, 6 μg/ml) for 24 h. Identical amounts of total RNA were analyzed by northern blot using a gene-specific DNA probe. As a control for loading, ethidium-bromide-stained, large rRNAs are shown.
Combination effects of MDM2 inhibition and CPT-induced DNA damage
It could be anticipated that drugs that damage the DNA in the cell or otherwise activate the p53 response could act in synergy with antisense agents that activates p53 by reducing MDM2 levels, as has previously been indicated employing phosphorothioates (24). Thus the ability of T5-12–Acr to enhance DNA-damage induced activation of p53 and cell death was studied using the topoisomerase I inhibitor anticancer agent CPT (Figure 9). Transcriptional activation by p53 was measured as the increase of the ratio of firefly luciferase activity (reporter)/Renilla luciferase activity (control) (vide supra) in JAR cells transfected with pRL-CMV control vector and pGL3-53RE reporter vector and treated with a combination of CPT and T5-12–Acr /LFA. A clear, dose-dependent combination effect was observed (Figure 9A) resulting in up to 38-fold induction of p53 activity at 2.5 μM CPT and 1 μM T5-12–Acr over control in the JAR cells. Incubation with 1 μM T5-12–Acr alone resulted in only a 5-fold activation of the p53 activity and CPT (2.5 μM) alone activated the p53-induced transcription only 2.5-fold. Finally the mismatch PNA (T5-12-mm–Acr) induced p53 activities up to 2-fold and only up to 6-fold in combination with 2.5 μM of CPT, again strongly supporting a sequence-specific antisense mechanism of the effects observed. This would indicate a synergy between the PNA action and CPT.
Figure 9. Effect of the T5-12–Acr in combination with camptothecin (CPT) on the cellular viabilities and p53-activation. (A) Transcriptional activation of p53 induced by T5-12–Acr and CPT in JAR cells. Cells were transfected with the p53-inducible firefly luciferase vector (pGL3-53RE), control Renilla luciferase vector (pRL-CMV), CPT, PNAs (1 μM) and LFA (6 μg/ml) simultaneously for 24 h. T5-12-mm–Acr was used as a 2 base pair mismatch control. Transcriptional activation by p53 is shown as the ratio of firefly luciferase activity/Renilla luciferase activity. Data are mean ± SD of four independent experiments. (B) Inhibition of cell growth by T5-12–Acr in combination with CPT. JAR cells were incubated with CPT and Acr–PNAs (2 μM) and LFA (6 μg/μl) for 24 h. Cellular viabilities were determined by an MTS assay (Promega). Data are mean ± SD of four independent experiments.
To study if the effects of T5-12–Acr with CPT are also reflected phenotypically, the cellular growth inhibition was assayed. Although the differential effect was not as pronounced as that observed on p53 activity, the cells treated with CPT (2.5 μM) in combination with T5-12–Acr showed only a 23% cell survival compared to 70% upon treatment with CPT only (Figure 9B). Again the mismatch control T5-12-mm–Acr did not induce any significant additional cell death in combination with CTP.
CONCLUSIONS
We have demonstrated that it is possible to elicit antisense effects in human tumor cell lines using Acr-conjugated PNA oligomers delivered via LFA-mediated transfection. Our results indicate that this novel delivery method functions in both JAR and HeLa cells. Further studies will reveal the generality and efficacy of the method in terms of other cell types.
We ascribe the observed effects of T5-12–Acr on JAR cells to an antisense mechanism (mismatch control PNA showed no effect under similar conditions) by which the PNA upon binding the target on the mdm2 mRNA inhibits translation. This conclusion is consistent with the finding that the MDM2 protein levels are reduced and p53 levels and activity are increased while mdm2 mRNA levels are unchanged. Finally, this PNA was cytotoxic to JAR cells, and it enhanced the cytotoxicity of CPT. Therefore, this PNA could constitute a lead for further developments (e.g. in mouse xenograft cancer models) of PNA-based anti-mdm2 anticancer drugs.
ACKNOWLEDGEMENTS
This study was supported by the Danish Cancer Society, the Lundbeck Foundation (Senior research fellowship to T.S.) and the Danish Medical Research Council.
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* To whom correspondence should be addressed. Tel: +45 35327762; Fax: +45 35396042; Email: pen@imbg.ku.dk
ABSTRACT
A series of peptide nucleic acid (PNA) oligomers targeting the mdm2 oncogene mRNA has been tested for the ability to inhibit the growth of JAR cells. The effect of these PNAs on the cells was also reflected in reduced levels of the MDM2 protein and increased levels of the p53 tumor suppressor protein, which is negatively regulated by MDM2. Initially, PNA oligomers were delivered as DNA complexes with lipofectamine, but it was discovered that PNA conjugated to the DNA intercalator 9-aminoacridine (Acr) (Acr–PNA) could be effectively delivered to JAR cells (as well as to HeLa pLuc705 cells) even in the absence of a DNA carrier. Using such lipofectamine-delivered Acr–PNA conjugates, one PNA targeting a cryptic AUG initiation site was identified that at a concentration of 2 μM caused a reduction of MDM2 levels to 20% (but no reduction in mdm2 mRNA levels) and a 3-fold increase in p53 levels, whereas a 2-base mismatch control had no such effects. Furthermore, transcriptional activation by p53 was also increased (6-fold), and cell viability was reduced to 80%. Finally, this PNA acted cooperatively with camptothecin treatment both with regard to p53 activity induction as well as cell viability. Using this novel cell delivery system, we have identified a target on the mdm2 mRNA that appears sensitive to antisense inhibition by PNA and therefore could be used as a lead for further development of mdm2-targeted antisense (PNA and other) gene therapeutic anticancer drugs.
INTRODUCTION
The potential of peptide nucleic acid (PNA) in anticancer drug discovery is still rather poorly explored. This is to a large extent due to the lack of a simple and robust method for the delivery of PNA to eukaryotic cells. Nonetheless, encouraging results have been reported in a range of systems (1–5). A number of studies have exploited cell-penetrating peptides as carriers of PNA (1,6–8), but (at least some of) these predominantly deliver PNA to the endosomal (lysosomal) compartment of the cell and not very efficiently to the cytosolic or nuclear compartment where the agent should exert its antisense action. Furthermore, most PNA–peptide conjugates significantly complicate the synthetic availability of these agents. Antisense oligonucleotides are effectively delivered to eukaryotic cells via cationic liposome complexation (9). Although PNA oligomers being molecules with a neutral charge cannot directly complex with (cationic) liposomes, delivery via this route is possible using PNA–lipid conjugates (10,11) or PNA–DNA hybrid complexes (12,13). In particular, the latter method appears general and effective, although the need for specifically optimized DNA oligonucleotides for each new PNA oligomer is a cumbersome drawback. Nevertheless, we decided to exploit this system in an anticancer drug discovery approach using the oncogene mdm2 as target.
The MDM2 protein is over-expressed and/or up-regulated in 40–60% of human sarcomas as well as in several solid tumors which may have a wild-type p53 (14–17). The regulation of the expression of MDM2 and p53 is tightly coupled as the expression of MDM2 is induced by p53 (18,19). Furthermore, MDM2 binds to p53 with a high affinity, inhibiting its ability to act as a transcription factor and also induces its degradation (20,21), indicating that MDM2 functions as a negative feed back regulator of p53 (21). Therefore, MDM2 is recognized as an important target for cancer therapy (22,23), resulting in up-regulation of p53 (20,24,25). In agreement with this hypothesis, an mdm2 antisense phosphorothioate oligonucleotide (PS) was shown to reduce MDM2 expression and to release p53 from a p53–MDM2 complex in a variety of cancer cell lines (20,24–26).
Furthermore, the mdm2 gene apart from being a relevant anticancer drug target, offers the possibility of effect analysis on both MDM2 down-regulation and p53 up-regulation. Finally, synergy on cell survival and p53 activity would be expected with traditional DNA damaging (e.g. via topoisomerase interference) anticancer drugs.
In contrast to (phosphorothioate) oligonucleotides, PNA oligomers do not activate RNaseH upon binding to mRNA. Consequently, sequence targets on a given mRNA that are sensitive to RNaseH-recruiting antisense agents are not necessarily good targets for PNA antisense agents. Thus the objectives of this study were to identify mdm2 mRNA sequence targets that are sensitive to PNA antisense agents. As a parallel objective we also seek to discover novel delivery methods for PNA oligomers.
MATERIALS AND METHODS
PNA synthesis
The sequences of the PNAs targeting mdm2 mRNA are shown in Figure 1. For the splicing correction study with HeLa pLuc705 cells, two 9-aminoacridine (Acr)–PNAs were used. PNA synthesis was carried out as reported previously (27). High-performance liquid chromatography (HPLC) analysis was performed on Delta Pak C18 column (5 μm, 3.9 x 150 mm) at room temperature with a flow rate of 1 ml/min using an acetonitrile/water (containing 0.1% TFA) gradient. The purification was performed on a C18 (Delta Pak, Waters, Copenhagen, Denmark) (15 μm, 19 x 300 mm) with a flow rate of 8 ml/min. All compounds were characterized by mass spectrometry using a Kratos MALDI-II time-of flight mass spectrometer and 3,5-dimethoxy-4-hydroxycinnamic acid as matrix. The PNAs were lyophilized and stored at 4°C. SV40 core nuclear localization signal (NLS) (PKKKRKV) was covalently linked to PNA at the N-terminal through an ethyleneglycol linker (eg1, 8-amino-2,6-dioxaoctanoic acid) via continuous synthesis. N-(9-aminoaciridinyl)-6-aminohexanoic acid (Acr) was linked to PNA on the solid support at the N-terminal of the PNA (28) through the eg1 linker (Acr–PNA). Fluorescein (Fl)-labeled PNA oligomers were prepared using a previously described ‘Fl–lysine’ monomer (29).
Figure 1. Sequences and location of the PNAs within the sequence of MDM2 mRNA (accession no. NM_002392 , 272–512 bp). (A) Target positions of PNAs are indicated as T1, T2, T3, T4 and T5. Arrows indicate the location and length of the antisense PNAs. (B) T5-12-mm and T5-12-mm–Acr are the mismatch PNA of T5-12 and T5-12–Acr respectively. eg1, 8-amino-2,6-dioxaoctanoic acid; Fl, fluorescein. PNA No.(1) indicates nomenclatures of the PNAs used in this study. PNA No.(2) indicates the reference PNA numbers. Unmodified PNAs are indicated in plain text. 9-aminoacridine (Acr)-conjugated PNAs (Acr–PNAs) are indicated with underlines. NLS-PNAs, PNAs conjugated with the SV40 core NLS (PKKKRKV), are indicated in italic.
Cell cultures
JAR cells were a kind gift from Dr Peter Ebbesen (The Stem Cell Laboratory, Aalborg University, Denmark). HeLa pLuc705 cells (29) were purchased from Gene Tools (USA). Both cell lines were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% GLUTAMAX I (GIBCO, Invitrogen). All cell cultures were incubated at 37°C with 5% CO2 for a minimum of 16 h before initiating transfection.
PNA transfection
Cells were plated and incubated at 37°C to give 40–60% confluence at the time of transfection. Before the transfection, the culture medium was replaced with OPTI-MEM (1% FBS) (Life Technologies) of sufficient volume to cover the cells. For the transfection of PNAs by the complementary DNA and LipofectAMINE (LFA, Life Technologies), the method of Doyle et al. (13) was used. Oligo-DNAs of the same length and complementary sequence to the corresponding PNAs were used in this study.
For Acr–PNA transfection, Acr–PNA/calf thymus DNA (CT-DNA)/LFA complex was prepared in OPTI-MEM. Five microliters of Acr–PNA (200 μM for a final concentration of 2 μM in the medium) were mixed with 40 μl of OPTI-MEM and incubated for 35 min at room temperature. The solution was mixed with 50 μl of diluted LFA solution (diluted with OPTI-MEM to 60 and 80 μg/ml respectively for JAR cells and HeLa pLuc705 cells) and incubated for 35 min at room temperature unless otherwise stated. This solution was added to the cells in 0.4 ml of OPTI-MEM (1% FBS) and incubated under the normal cell growth condition.
Cell proliferation assay
Cells were plated in 96-well microtiter plates (Nunc) and grown to 40% cell confluence at the time of transfection. PNA/complementary-DNA/LFA complex was transfected at the indicated concentrations as mentioned above. The camptothecin (CPT) solution (when desired) was added to the cultured cells at the indicated concentration simultaneously with the transfection of Acr–PNA/LFA complex as mentioned above. Cell viabilities were assayed using the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Bie&Berntsen A/S, Denmark) according to the manufacturer's instructions.
Western blot analysis
Cells were lysed in boiling lysis buffer (1% SDS, 10 mM Tris–HCl, pH 7.2, 1x protease inhibitor cocktail tablet (Roche, Germany)], and 40 μg of the protein lysate was fractionated by SDS–PAGE and transferred to nitrocellulose membranes (ADVANTEC MFS). The nitrocellulose membrane was incubated with blocking buffer (2% BSA, 20 mM Tris–HCl, pH 7.2, 150 mM NaCl and 0.01% Tween-20) overnight at 4°C. The membrane was incubated with primary antibody overnight at 4°C. The membrane was washed with washing buffer (20 mM Tris–HCl, pH 7.2, 150 mM NaCl and 0.01% Tween-20) three times for a minimum of 15 min each at room temperature, and then incubated with 1:20 000 diluted anti-mouse IgG–alkaline phosphate-conjugated secondary antibody (Sigma) for 1 h at room temperature. After washing as described above, the protein of interest was detected with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (BCIP).
Fluorescence microscopy
For uptake analysis, exponentially growing cells were plated in 4-well chamber slides (Nunc) the day before transfection. Transfections were performed as described above using Fl-labeled PNAs (PNA2297, 2415) with the LFA (6 and 8 μg/ml for JAR cells and HeLa pLuc705 cells, respectively). After 16 h, the cells were rinsed twice with growth medium (RPMI 1640 containing 10% FBS and 1.5% glutamax) and subjected to microscopic studies without fixation. For the propidium iodide counter staining, cells were incubated with 8 μg/ml of propidium iodide for 15 min at 37°C before being subjected to the microscopic study. Cells were visualized using a LEITZ WETZLAR (Germany) fluorescent microscope (DIALUX 22EB) at 400 x magnification with filter block N2 (Ernst Leitz Wetzlar GmbH, Germany).
Luciferase assay
Exponentially growing HeLa pLuc705 cells were plated in 24-well plates the day before transfection. For the transfection, Acr–PNA (PNA2391), targeting the aberrant splice site within the luciferase pre-mRNA was used in combination with LFA (8 μg/ml) and CT-DNA as described above (see the PNA synthesis for the sequences of PNA2391 and PNA2746). After 24 h, cells were lysed with Passive Lysis Buffer (Promega) and subjected to luciferase activity measurements. Luciferase activities were measured with Luminoskan TL Plus (Thermo Labsystems) by using the Luciferase Assay System (Promega) following the manufacturer's procedures. The relative light units (RLU) were normalized to micrograms of protein determined by the Bradford method (Coomassie Plus, Pierce) following the manufacturer's procedure.
RT–PCR
Exponentially growing HeLa pLuc705 cells were treated with Acr–PNA/LFA as described in the luciferase assay section. Total RNA was extracted from the cells by using RNeasy Mini kit (Qiagen) and subjected to RT–PCR. RT–PCR was performed by using OneStep RT–PCR kit (Qiagen). Three nanograms of total RNA was used for each RT–PCR reaction (20 μl). Primers for RT–PCR were as follows: forward primer, 5'-TTGATATGTGGATTTCGAGTCGTC-3'; reverse primer, 5'-TGTCAATCAGAGTGCTTTTGGCG-3'. RT–PCR program was as follows: 55°C for 35 min, 1 cycle; 95°C for 15 min, 1 cycle; 94°C for 0.5 min, 55°C for 0.5 min, 72°C for 0.5 min, 30 cycles]. RT–PCR products were analyzed on a 2% agarose gel run in 1x TBE buffer and visualized by ethidium bromide staining. Gel images were captured by ImageMaster (Pharmacia Biotech) and analyzed by UN-SCAN-IT software (Silk Scientific Corporation).
Northern analysis
For northern blot analysis, 15 μg of total RNA was analyzed using AlkPhos DIRECT (Amersham Pharmacia Biotech) according to the manufacturer's instructions. A double-stranded DNA fragment of MDM2, bases 312–1787 (GenBank accession no. NM_002392 ), were used as a gene specific probe.
DNA damage treatment and luciferase assay
A firefly luciferase vector holding the p53-responsive element of human MDM2 upstream of the firefly luciferase gene, was used as a reporter vector (pGL3-53RE). The pRL-CMV vector (Promega), holding a Renilla luciferase, was used as a control vector. These two plasmid DNAs were mixed (pGL3-53RE:pRL-CMV of 49:1) and used for transfection. Transcriptional activation by p53 was measured as the ratio of firefly luciferase activity (reporter)/Renilla luciferase activity (control). For construction of the reporter vector pGL3-53RE, p53-responsive element sequence (GenBank/EMBL accession no. AF144015 , nucleotides 95–179) from human MDM2 gene was cloned into the pGL3-Basic Vector (Promega) upstream of the luc+ gene (18).
For the DNA damage treatment, CPT was added to the cultured cells at the indicated concentration simultaneously with the addition of Acr–PNA/LFA mixture as mentioned above. The luciferase activities were determined by Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions.
RESULTS AND DISCUSSION
Initially, we designed a series of PNA oligomers targeting the translation initiation site and regions immediately downstream from this on the mdm2 mRNA. We also included an internal ATG site . All of these PNAs were also tested as conjugates with the NLS peptide (PKKKRKV) as this has been reported to enhance cellular delivery (1,28), and at least should ensure nuclear localization of the PNA. All PNAs were delivered to the JAR cells at 1 μM concentration using the oligonucleotide–LFA protocol described by Doyle et al. (13).
A cell viability screening revealed (Figure 2) that only three of the PNAs (T1-12, T5-12 and T5-12-NLS) showed cellular cytotoxicity as an indication of antisense activity. We therefore decided to examine these PNAs (as well as the NLS version of T1-12:T1-12-NLS) further in terms of the effects on MDM2 and p53 expression. The results fully support the contention that the effects on cell viability are indeed caused by antisense down-regulation of MDM2 and a concomitant up-regulation of p53 (Figure 3A) since PNAs T1-12, T5-12 and T5-12-NLS show clear effects on protein levels. It is noteworthy that conjugation of NLS to the PNA did not significantly enhance the antisense effect. Indeed, for the T1 target, conjugation to NLS decreased the effect. Furthermore, T5-12-NLS showed no effects on cell viability up to 10 μM when delivered to the cells without the carrier oligonucleotide and LFA (data not shown) indicating that the NLS peptide is not capable of delivering this PNA oligomer to JAR cells. This is in contrast to results reported in Burkitt's lymphomas (BL) cells (1).
Figure 2. Effect of target site on the cellular viabilities by antisense PNAs. PNAs (final concentration of 1 μM) were transfected to JAR cells as complexes with complementary DNA and LFA (6 μg/ml) (2). After 18 h of transfection, cell viabilities were determined by MTS assay (Promega).
Figure 3. Effect of antisense-PNAs on MDM2 and p53 levels in JAR cells. Cells were transfected with PNA as complexes with complementary DNA and LFA for 24 h. Identical amounts of protein were analyzed by western blot using monoclonal anti MDM antibody and anti p53 antibody. (A) T1-12, T1-12-NLS, T5-12 and T5-12-NLS were used at 1 μM. (B) T5-12 was tested at 0.5, 1 and 2 μM. Mismatch PNA (T5-12-mm), holding 2 bp mismatches, was used at 2 μM.
In order to further substantiate an antisense mechanism, we tested a double mismatch (base interchange) derivative of the active T5-12 (T5-12-mm) and this showed no effect on either the MDM2 or the p53 levels at 2 μM, at which concentration T5-12 reduced the MDM2 level to 15% and resulted in a 2.4-fold increase in p53 level (Figure 3B). Finally, we compared the dose response of T5-12 and T5-12-mm cell survival assay (Figure 4A), in which T5-12 showed cytotoxicity <1 μM, whereas T5-12-mm did not affect cell viability up to 5 μM.
Figure 4. Effect of the T5-12 on the cellular viabilities and p53-activation. (A) JAR cells were transfected with T5-12 and T5-12-mm (mismatch control) at the indicated concentrations for 36 h as complexes with complementary DNA and LFA. Cellular viabilities were determined by MTS assay (Promega). Data are mean ± SD of six independent experiments. The value without PNA was set as 100% survival. (B) Transcriptional activation of p53 induced by T5-12 in JAR cells. Cells were transfected with the PNA/complementary-DNA/LFA complex (1 μM PNA) for 3 h followed by a transfection of vectors (p53-inducible firefly luciferase vector (pGL3-53RE) and pRL-CMV vector as an internal control) as a complex with LFA (6 μg/ml) for 24 h. T5-12-mm was used as a 2 bp mismatch control. Transcriptional activation of p53 is shown as a ratio of firefly luciferase activity/Renilla luciferase activity. Data are mean ± SD of three independent experiments.
In order to study whether the increased level of p53 was also translated into increased p53 activity, we employed a dual luciferase reporter assay in which one vector harbors the firefly luciferase gene controlled by the p53 responsive element at the promoter and the Renilla luciferase controlled by the constitutive cytomegalovirus (CMV) promoter is used as internal control. Thus the ratio of firefly versus Renilla luciferase activity will be a measure of p53 activity. As shown by the results presented in Figure 4B, treatment of the cells with PNA T5-12 does indeed result in a relative increase in firefly luciferase activity as compared with non-treated controls cells or to cells treated with the double mismatched T5-12-mm, which in this assay showed some activation of p53, as would be expected from the p53 levels (Figure 3B).
Therefore, using the oligonucleotide–LFA delivery method, we have identified a possible PNA anticancer drug antisense lead target (that of T5-12) on the mdm2 mRNA. However, this delivery method is not adequate for screening of large numbers of PNA oligomers. Using PNA T5-12 as the lead compound, we decided to explore an alternative delivery method. We reasoned that a PNA oligomer conjugated to the DNA intercalator Acr should confer general affinity for double-stranded DNA, and that using such acridine–PNA conjugates could allow the use of, e.g., CT-DNA as a general carrier in combination with cationic lipids. Consequently, we synthesized a T5-12–Acr conjugate (PNA2296) and a similar compound also containing Fl (T5-12–Acr–Fl, PNA2297) (Figure 5A).
Figure 5. Structures and transfection studies of Acr-conjugated PNAs. (A) Chemical structures of 9-aminocaprorylacidine (Acr)-conjugated PNA , Fl-labeled Acr–PNA and Fl-labeled PNA . (B) Transfection studies of Acr–PNA with JAR cells and HeLa pLuc705 cells. T5-12–Acr–Fl (1 μM) (Acr–Fl–PNA, Fl-labeled Acr–PNA) or T5-12–Fl (1 μM) (Fl–PNA, Fl-labeled PNA without Acr) was transfected into the cells for 16 h in combinations with or without CT-DNA (1 μg/ml) and/or LFA (6 and 8 μg/ml for JAR cells and HeLa pLuc705 cells, respectively). Cells were incubated with Acr–Fl–PNA/CT-DNA/LFA (a), Acr–Fl–PNA (b), Acr–Fl–PNA/CT-DNA (c), Acr–Fl–PNA/LFA (d) or Fl–PNA/LFA (e). (C) Acr–Fl–PNA transfection study of JAR cells and HeLa pLuc705 cells with propidium iodide counter staining. Transfection of the Acr–Fl–PNA were performed under the same condition in (B)–(d). Cells were treated with 8 μg/ml of propidium iodide for 15 min before being subjected to microscopic study.
Cellular uptake of Acr–PNA
Transfection of Fl-labeled Acr-conjugated PNAs (Acr–Fl–PNA, T5-12–Acr–Fl) were tested in combination with CT-DNA and LFA (Figure 5B). Transfection with the Acr–Fl–PNA/CT-DNA/LFA complex resulted in a high level of fluorescence in treated JAR cells and HeLa pLuc705 cells . As a control experiment, Acr–Fl–PNA was added to the cells either free or in complex with CT-DNA and/or LFA. Unexpectedly, the transfection of the cells by Acr–Fl–PNA in combination with LFA also resulted in a high level of fluorescence in the treated cells without showing severe cellular toxicity (counter staining with propidium iodide in Figure 5C). However, treatment with Acr–Fl–PNA alone or Acr–Fl–PNA in combination with CT-DNA did not result in any significant intracellular fluorescence . In addition, Fl-labeled PNA (Fl–PNA, T5-12–Fl), not containing the Acr, was not effectively delivered by LFA either to JAR cells or to HeLa pLuc705 cells . Thus, inclusion of DNA was not required for effective cellular uptake. Although the fluorescence microscopy data do not allow a detailed determination of the intracellular localization of the Acr–PNA, most of the PNA does appear to be present in vesicles (endosomes?) and not evenly distributed in the cytoplasm. No preferential staining of the nucleus is observed either. Furthermore, a counterstain with propidium iodide which stains only dead cells clearly indicates that cells that take up the PNA are still alive and healthy. We believe that the aminoacridine on the non-protonated form in analogy to fatty acids (10) is sufficiently lipophilic to allow complexation with the liposomes, whereas the protonated form increases solubility in the aqueous environment. (If this is indeed the mechanism, it should be possible to further develop this concept using other analogous polycyclic ligands.)
To support the conclusion of effective cellular internalization of the Acr–PNA by LFA delivery, we employed the sensitive luciferase antisense activation method with HeLa pLuc705 cells developed by Kole et al. (30). HeLa pLuc705 cells are stably transfected HeLa cells with a modified luciferase containing the mutated intron2 of the human ?-globin pre-mRNA causing pre-mRNA missplicing and resulting in a non-functional enzyme. By using PNA targeting the aberrant splice site, this assay can measure antisense activity (reflecting effective uptake) of PNA as restored luciferase activity as a consequence of mRNA splice correction by the PNA (30).
We chose a PNA sequence previously identified by Kole et al. (30,31). As anticipated the corresponding Acr–PNA (PNA2391) showed good antisense activity (as luciferase activity) when complexed with LFA or LFA/CT-DNA but showed no significant activity without LFA (Figure 6A). These data fully corroborate the uptake studies (Figure 5B). In addition, the cells transfected with Acr–PNA/LFA showed comparable antisense activity as compared with the conventional method, using complexes of PNA/complementary-DNA/LFA (data not shown). However, a more systematic comparative study in more systems (cell types and gene targets) are required in order to draw more general quantitative conclusions of the relative efficacy of the two delivery methods. The antisense effect of the Acr–PNA was also demonstrated by a dose-dependent correction of missplicing (Figure 6B and C) (47% at 4 μM of Acr–PNA), which correlated fully with the increase of luciferase activity. Finally, a control mismatch PNA (MM, PNA2746) did not show (antisense) activity either in the presence or in the absence of LFA. These results demonstrate that the Acr–PNAs, transfected as a complex with LFA, are to a significant extent delivered into the nucleus where the Acr–PNA binds the target mRNA and corrects splicing.
Figure 6. Acr–PNA transfection studies with the HeLa pLuc705 cells. HeLa pLuc705 cells were transfected with Acr–PNA (PNA 2391), targeting the cryptic splice site of luciferase pre-mRNA to correct the missplicing, or its mismatch PNA (MM, PNA2746) at the indicated concentrations in combination with or without CT-DNA (1 μg/ml) and/or LFA (8 μg/ml). After 24 h, cells were subjected to the further analysis. Luciferase activities were measured by Luciferase Assay System (Promega). The obtained RLU were normalized to micrograms of protein. Each data point is an average of three independent experiments ± SD. (A) Acr–PNAs (1 μM) with/without addition of LFA and/or CT-DNA were transfected for 24 h. (B) The cells were treated with PNA2391 (Acr–PNA) or mismatch PNA (MM, PNA2746) in the absence (–LFA) or presence of LFA (+LFA) for 24 h. Total RNA was extracted from the cells and subjected to RT–PCR analysis. U, uncorrected form (268 bp), which contains aberrant exon. C, corrected form (142 bp) in which the entire intron was eliminated. The values under the lower panel (+LFA) indicate the relative amount of corrected form. The corrected form in LFA-free transfections amounted to <1% in all cases. (C) HeLa pLuc705 cells were transfected as in (B) and the cells were subjected to a luciferase activity analysis.
Thus the LFA/Acr–PNA delivery method is a convenient method for testing larger series of PNA oligomers, as no (sequence designed) carrier oligonucleotide is required. Consequently, we used Acr–PNA/LFA transfection for further experiments.
Inhibition of MDM2 expression by antisense Acr–PNAs
A series Acr–PNAs targeted within the mdm2 mRNA were tested at a concentration of 1 μM for their ability to down-regulate MDM2 (Figure 7). Among the six Acr–PNAs (12 bp of each) tested, T2-12–Acr and T5-12–Acr showed significant down-regulation of the MDM2 protein level to 33 and 27% of the control, respectively (Figure 7A).
Figure 7. Inhibition of MDM2 protein expression by antisense Acr–PNAs. JAR cells were treated by PNAs with LFA (6 μg/ml) for 24 h. Identical amounts of protein were analyzed by western blot using a monoclonal anti-MDM antibody and/or monoclonal anti-p53 antibody. (A) Six Acr–PNAs (1 μM) of 12 bp targeted to the different position within the mdm2 mRNA sequence were tested for down-regulation of MDM2 expression. (B) The effect of Acr–PNA length on the antisense effects. Acr–PNAs (1 μM) with different length (8, 10, 12, 14, 16 and 18 bp) were tested for down-regulation of MDM2 expressions. (C) The effect of Acr–PNA length on the MDM2 and p53 expression levels in JAR cells. Seven Acr–PNAs (1 μM) with different lengths (12, 13, 14, 15, 16, 18 and 20 bp) were tested. (D) The effect of T5-12–Acr on the MDM2 and p53 expression in JAR cells. T5-12–Acr (Acr–PNA), T5-12-mm–Acr (mismatch control for T5-12–Acr) and T5-12 (naked PNA) were used at the concentrations indicated.
Using these two PNAs as leads, a series of Acr–PNAs of different length from 8 bp to 18 bp were also tested (Figure 7B and C). These results revealed a clear optimum length of 12–16 bases for the T2-12–Acr series and an optimum around 13 bases for the T5-12–Acr series assayed both for MDM2 reduction as well as p53 increase (Figure 7C). In contrast to the previous report by Corey's group (32) about the requirements of the longer PNA (>17 bp) for the antisense effects, the longer PNAs (18–20 bases) showed a weaker effect than the shorter PNAs. However, it should be emphasized that in these experiments we were not able to separate the differences in the efficiency of delivery and antisense activity, as the results reflect the effect of the treatment as such, and the weaker effect of the longer PNAs could be due to a less effective delivery. This phenomenon was previously observed in bacteria (33).
It is noteworthy, that the most active of the Acr–PNAs, T5-12–Acr, is an acridine conjugate of the most active unmodified PNA (T5-12), thereby supporting the sensitivity of this sequence target on the mdm2 mRNA. Thus, we decided to study this PNA further. First, we wished to obtain evidence that the observed MDM2 down-regulation was accomplished through a sequence-specific antisense mechanism by using a mismatch PNA (T5-12-mm–Acr) as a control (Figure 7D). This experiment showed that while fully matched PNA (T5-12–Acr) down-regulates MDM2 in a dose-dependent manner, mismatched PNA (T5-12-mm–Acr) containing two mismatches did not significantly inhibit MDM2 expression even at a concentration of 2 μM. As a further control of the delivery system T5-12 lacking the Acr did not show significant down-regulation of MDM2 under these conditions. As expected the p53 levels showed opposite tendencies as compared with the MDM2 protein levels. T5-12–Acr caused a 2.9-fold up-regulation of p53 at 2 μM, while none of the control PNAs (T5-12-mm–Acr and T5-12) had such effects. Finally, the effect on mdm2 mRNA levels was assayed by northern blot analysis, and no detectable change upon treatment with T5-12–Acr was observed (Figure 8). Thus the mechanism appears to be translation inhibition rather than mRNA destabilization. This is somewhat surprising in view of the position of the target 150 bases downstream of the translation initiation, which is usually considered more sensitive for non-RnaseH-supported antisense inhibition. The target is at a cryptic AUG initiation site, but the function of this is obscure. Thus the molecular mechanism by which T5-12–Acr and T5-12 (and analogues) inhibit MDM2 translation is not clear at this stage, but most likely involves translation elongation arrest, as previously observed in some cases (34).
Figure 8. Northern blot analysis of mdm2 mRNA expression in JAR cells treated with Acr–PNAs (2 μM) with LipofectAMINE (LFA, 6 μg/ml) for 24 h. Identical amounts of total RNA were analyzed by northern blot using a gene-specific DNA probe. As a control for loading, ethidium-bromide-stained, large rRNAs are shown.
Combination effects of MDM2 inhibition and CPT-induced DNA damage
It could be anticipated that drugs that damage the DNA in the cell or otherwise activate the p53 response could act in synergy with antisense agents that activates p53 by reducing MDM2 levels, as has previously been indicated employing phosphorothioates (24). Thus the ability of T5-12–Acr to enhance DNA-damage induced activation of p53 and cell death was studied using the topoisomerase I inhibitor anticancer agent CPT (Figure 9). Transcriptional activation by p53 was measured as the increase of the ratio of firefly luciferase activity (reporter)/Renilla luciferase activity (control) (vide supra) in JAR cells transfected with pRL-CMV control vector and pGL3-53RE reporter vector and treated with a combination of CPT and T5-12–Acr /LFA. A clear, dose-dependent combination effect was observed (Figure 9A) resulting in up to 38-fold induction of p53 activity at 2.5 μM CPT and 1 μM T5-12–Acr over control in the JAR cells. Incubation with 1 μM T5-12–Acr alone resulted in only a 5-fold activation of the p53 activity and CPT (2.5 μM) alone activated the p53-induced transcription only 2.5-fold. Finally the mismatch PNA (T5-12-mm–Acr) induced p53 activities up to 2-fold and only up to 6-fold in combination with 2.5 μM of CPT, again strongly supporting a sequence-specific antisense mechanism of the effects observed. This would indicate a synergy between the PNA action and CPT.
Figure 9. Effect of the T5-12–Acr in combination with camptothecin (CPT) on the cellular viabilities and p53-activation. (A) Transcriptional activation of p53 induced by T5-12–Acr and CPT in JAR cells. Cells were transfected with the p53-inducible firefly luciferase vector (pGL3-53RE), control Renilla luciferase vector (pRL-CMV), CPT, PNAs (1 μM) and LFA (6 μg/ml) simultaneously for 24 h. T5-12-mm–Acr was used as a 2 base pair mismatch control. Transcriptional activation by p53 is shown as the ratio of firefly luciferase activity/Renilla luciferase activity. Data are mean ± SD of four independent experiments. (B) Inhibition of cell growth by T5-12–Acr in combination with CPT. JAR cells were incubated with CPT and Acr–PNAs (2 μM) and LFA (6 μg/μl) for 24 h. Cellular viabilities were determined by an MTS assay (Promega). Data are mean ± SD of four independent experiments.
To study if the effects of T5-12–Acr with CPT are also reflected phenotypically, the cellular growth inhibition was assayed. Although the differential effect was not as pronounced as that observed on p53 activity, the cells treated with CPT (2.5 μM) in combination with T5-12–Acr showed only a 23% cell survival compared to 70% upon treatment with CPT only (Figure 9B). Again the mismatch control T5-12-mm–Acr did not induce any significant additional cell death in combination with CTP.
CONCLUSIONS
We have demonstrated that it is possible to elicit antisense effects in human tumor cell lines using Acr-conjugated PNA oligomers delivered via LFA-mediated transfection. Our results indicate that this novel delivery method functions in both JAR and HeLa cells. Further studies will reveal the generality and efficacy of the method in terms of other cell types.
We ascribe the observed effects of T5-12–Acr on JAR cells to an antisense mechanism (mismatch control PNA showed no effect under similar conditions) by which the PNA upon binding the target on the mdm2 mRNA inhibits translation. This conclusion is consistent with the finding that the MDM2 protein levels are reduced and p53 levels and activity are increased while mdm2 mRNA levels are unchanged. Finally, this PNA was cytotoxic to JAR cells, and it enhanced the cytotoxicity of CPT. Therefore, this PNA could constitute a lead for further developments (e.g. in mouse xenograft cancer models) of PNA-based anti-mdm2 anticancer drugs.
ACKNOWLEDGEMENTS
This study was supported by the Danish Cancer Society, the Lundbeck Foundation (Senior research fellowship to T.S.) and the Danish Medical Research Council.
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