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Mol Cancer Ther. 2006;5:1683-1692
© 2006 American Association for Cancer Research

Research Articles: Therapeutics

Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells

Valentina Rapozzi, Susanna Cogoi and Luigi E. Xodo

Department of Biomedical Sciences and Technologies, School of Medicine, University of Udine, Udine, Italy

Requests for reprints: Luigi E. Xodo, Department of Biomedical Sciences and Technologies, School of Medicine, P.le Kolbe 4, 33100 Udine, Italy. Phone: 39-432-494395; Fax: 39-432-494301. E-mail: lxodo{at}makek.dstb.uniud.it


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic myeloid leukemia (CML) develops when a hematopoietic stem cell acquires the Philadelphia chromosome carrying the BCR/ABL fusion gene. This gives the transformed cells a proliferative advantage over normal hematopoietic cells. Silencing the BCR/ABL oncogene by treatment with specific drugs remains an important therapeutic goal. In this work, we used locked nucleic acid (LNA)–modified oligonucleotides to silence BCR/ABL and reduce CML cell proliferation, as these oligonucleotides are resistant to nucleases and exhibit an exceptional affinity for cognate RNA. The anti-BCR/ABL oligonucleotides were designed as LNA-DNA gapmers, consisting of end blocks of 3/4 LNA monomers and a central DNA stretch of 13/14 deoxyribonucleotides. The gapmers were complementary to the b2a2 and b3a2 mRNA junctions with which they form hybrid duplexes that have melting temperatures of 79°C and 75°C, respectively, in a 20 mmol/L NaCl-buffered (pH 7.4) solution. Like DNA, the designed LNA-DNA gapmers were capable of activating RNase H and promote cleavage of the target b2a2 and b3a2 BCR/ABL mRNAs. The treatment of CML cells with junction-specific antisense gapmers resulted in a strong and specific reduction of the levels of BCR/ABL transcripts (~20% of control) and protein p210BCR/ABL (~30% of control). Moreover, the antisense oligonucleotides suppressed cell growth up to 40% of control and induced apoptosis, as indicated by the increase of caspase-3/7 activity in the treated cells. Finally, the b2a2-specific antisense gapmer used in combination with STI571 (imatinib mesylate), a tyrosine kinase inhibitor of p210BCR/ABL, produced an enhanced antiproliferative effect in KYO-1 cells, which compared with K562 cells are refractory to STI571. The data of this study support the application of BCR/ABL antisense LNA-DNA gapmers, used either alone or in combination with STI571, as potential antileukemic agents. [Mol Cancer Ther 2006;5(7):1683–92]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chronic myeloid leukemia (CML) is a disorder that develops when a hematopoietic stem cell acquires the Philadelphia chromosome carrying the chimeric BCR/ABL oncogene resulting from a chromosomal translocation: t(9;22) (1, 2). This abnormal gene is obtained from the fusion of parts of two normal genes: the ABL gene on chromosome 9 and the BCR gene on chromosome 22. Both genes are present in all tissues, and whereas the Abl protein is a non-receptor tyrosine kinase that has an important role in signal transduction and regulation of cell growth (3), little is known about the function of protein Bcr. The BCR/ABL transcript contains one of two possible junctions, designed as b2a2 and b3a2 (4). The two types of transcripts encode for an oncoprotein of 210 kDa (i.e., p210Bcr/Abl). The oncogenic potential of this protein resides in the fact that whereas the normal Abl protein is characterized by a regulated tyrosine kinase activity, the Bcr/Abl protein is constitutively activated, leading to a deregulated cell proliferation and a decreased apoptosis in response to mutagenic stimuli (5). As the BCR/ABL oncogene plays a critical role in the pathogenesis of leukemia, it is thought to be an attractive target for anticancer drugs. Antisense strategies aiming to suppress the expression of BCR/ABL in CML cells have received the attention of several research groups over the last decade. This is a promising approach, and several antisense oligonucleotides have already reached the stage of clinical trials. The Food and Drug Administration approved the first antisense oligonucleotide in 1998 (6). Although antisense thioated oligonucleotides combine many desired properties, like a high biostability, RNase H activation, and target specificity, they have a few disadvantages compared with phosphodiester analogues, such as a reduced binding affinity for the target RNA and nonspecific binding to proteins (7–9). To overcome these obstacles, a variety of chemically modified oligonucleotides have been proposed. DNA analogues showing interesting properties in terms of nuclease stability, target affinity, and low cytotoxicity are peptide nucleic acid (10–15), N5'/P5' phosphoramidites (16), morpholino phosphoramidites (17), and hexitol nucleic acids (18, 19). In addition, locked nucleic acids (LNA) have been proposed as antisense molecules of enhanced properties (20, 21). LNA is a ribonucleotide analogue containing a methylene linkage between 2'-oxygen and 4'-carbon of the ribose ring. This constraint locks the sugar in the C-3'-endo conformation with the result that LNA-containing oligonucleotides exhibit a very high affinity for cognate DNA and RNA sequences (22, 23). In addition, full LNA and mixed LNA-DNA oligonucleotides show high serum stability, up to 10-fold increase of the half-life in human serum compared with unmodified oligonucleotides (24–27). LNA-DNA gapmer oligonucleotides were reported to exhibit in vivo a potent antisense activity against the {delta}-opioid receptor with respect to the analogue antisense full DNA oligonucleotide (28). In this study, we explored the capacity of LNA-containing oligonucleotides to inhibit the expression of the BCR/ABL oncogene in leukemic cells. We designed LNA-DNA gapmers specific for the b2a2 and b3a2 BCR/ABL mRNA junctions (b3a2 LNA-DNA and b2a2 LNA-DNA; Fig. 1 ) and studied their effect in K562 and KYO-1 cells, in terms of gene suppression, inhibition of cell proliferation, stimulation of apoptosis, and sensitization to STI571 (imatinib mesylate).


Figure 1
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  		Figure 1. A, chemical structures of DNA and LNA nucleotides. B, sequences of the b2a2 and b3a2 BCR/ABL mRNA junctions and of the designed LNA-DNA gapmers. The underlined bases in bold are LNA monomers. For each junction, a control gapmer with the same base content of the antisense gapmer but with a different sequence has been synthesized.

 

   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
K562 (provided by Dr. M. Giunta, Polyclinic of Udine, Italy) and KYO-1 (provided by Dr. C.M. Broughton, Department of Haematology, University of Liverpool, United Kingdom) cells were maintained in exponential growth in RPMI 1640 containing 100 units/mL penicillin, 100 mg/mL streptomycin, 20 mmol/L L-glutamine, and 10% fetal bovine serum (Celbio, Milan, Italy). Fetal bovine serum was heat inactivated at 56°C for 20 minutes. Cell viability was measured by trypan blue exclusion and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, following standard procedures.

LNA-DNA Oligonucleotides
The LNA-DNA gapmers 5'-AGGGCTTTTGAACTCTGCTT (b3a2 LNA-DNA), 5'-F-AGGGCTTTTGAACTCTGCTT (b3a2 LNA-DNA-F), 5'-GAGTCTTGTGACATGTCCTT (b3a2 cLNA-DNA), 5'-GGGCTTCTTCCTTATTGATGG (b2a2 LNA-DNA), and 5'-GGCGTCTTTCCTATTTGTAGG (b2a2 cLNA-DNA) and the oligonucleotides 5-AAGCAGAGUUCAAAAGCCCU (b3a2 RNA), 5'-CCAUCAAUAAGGAAGAAGCCC (b2a2 RNA), 5'-AGGGCTTTTGAACTCTGCTTT (b3a2 DNA), and 5'-GGGCTTCTTCCTTATTGATGG (b2a2 DNA) were purchased from Proligo (Paris, France). The LNA-DNA gapmers complementary to the b2a2 and b3a2 BCR/ABL mRNA junctions were synthesized with end blocks of 3/4 LNA monomers (bases in bold and underlined) and a central unmodified DNA of 13/14 nucleotides. The lyophilized compounds were dissolved in DEPC water, and solutions of 100 µmol/L were stored at –80°C. The LNA-DNAs were heated at 95°C for 5 minutes and quenched in ice before being transferred in K562 and KYO1 cells. STI571, 4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[4-(3-pyridinyl)-2-pyrimidinyl]amino]-phenyl]benzamide methanesulfonate, was kindly provided by Novartis (Basel, Switzerland). Circular dichroism spectra were recorded on a JASCO J-600 spectropolarimeter. They were recorded in a 0.2-cm path length cuvette containing duplex solutions at the concentration 3 µmol/L. Each spectrum was recorded thrice, smoothed, and subtracted to the baseline.

Confocal Microscopy
KYO-1 and K562 cells (0.8 x 105 in 600 µL RPMI) were exposed for 5 hours to fluorescein-labeled b2a2 LNA-DNA gapmer. The cells were cytospinned on a glass slide and fixed with 3% paraformaldehyde in PBS for 20 minutes. After washing with 0.1 mol/L glycine, containing 0.02% sodium azide in PBS to remove paraformaldehyde and Triton X-100 (0.1% in PBS), the cells were incubated with 24 µg/mL propidium iodide and 0.4 mg/mL RNase A for 30 minutes at 37°C to stain the nuclei. The cells were analyzed using a Leica DM IRBE confocal imaging system.

RNA Isolation and cDNA Synthesis
Total RNA was isolated following a standard guanidinium thiocyanate/phenol/chloroform extraction (29), whereas the cDNA synthesis was carried out as previously described (10).

PCR
PCR was carried out in a final volume of 50 µL containing 5 µL of cDNA, heated at 95°C for 5 minutes, and 10 µL of BCR/ABL mix [5 µL of 10x Taq polymerase buffer (EuroClone, Wetherby, United Kingdom); 0.75 µL of 25 mmol/L MgCl2; 1 µL of 12.5 pmol/µL primer EA122 (5'-GTTTCAGAAGCTTCTCCCTG); 1 µL of 12.5 pmol/µL primer EA500 (5'-TGTGATTATAGCCTAAGACCCGGAG); 1 µL of 5 mmol/L deoxynucleotide triphosphates with equimolar amounts of dTTP, dCTP, dATP, and dGTP (EuroClone); 0.25 µL of 5 units/µL Taq DNA polymerase (EuroClone)]. Amplification was carried out on an automated DNA thermal cycler (Progene) as follows: 35 cycles of denaturation (94°C for 30 seconds), annealing (60°C for 30 seconds), and extension (72°C for 30 seconds). Competitive reverse transcription-PCR was done according to the method described by Moravcova et al. (30). As a loading control, we amplified a 128-bp DNA fragment of ABL, using primers ABL1A (5'-CCTCTCGCTGGACCCAGTGA) and EA500 (annealing at 55°C, 35 cycles) as well as a 334-bp DNA fragment of glyceraldehyde-3-phosphate dehydrogenase, using primers Gsx (5'-AGTATGACAACAGCCTCAAG) and Gdx (5'-TTTTCTAGACGGCAGGTCAG; annealing at 50°C, 25 cycles).

Western Blotting
We measured the levels of ß-actin and protein Bcr/Abl in each cell lysates following a double-antibody procedure as previously described (10). We used c-ABL (Ab-3, 1:40, Calbiochem, La Jolla, CA) and ß-actin (1:5,000; Calbiochem) as primary antibodies, and horseradish peroxidase–conjugated anti-mouse IgG (1:10,000; Calbiochem) for Ab-3 and anti-mouse IgM (1:10,000; Calbiochem) for the ß-actin as secondary antibodies. Chemiluminescence was detected immediately as described by the manufacturer (Super Signal West Pico, Pierce, Rockford, IL). Films were exposed for about 5 minutes for Bcr/Abl and 1 minute for ß-actin. Protein levels were quantified by a densitometer (LKB Ultrascan XL Enhanced Laser Densitometer, Bromma, Sweden).

RNase H Assay
b2a2 or b3a2 RNA (30 pmol) were end labeled with 10 units T4 polynucleotide kinase (BioLabs, Hitchin, United Kingdom) with 0.37 MBq [{gamma}-32P]ATP (Amersham, Arlington Heights, IL) for 1 hour and 30 minutes at 37°C. Annealing between radiolabeled RNA targets (750 nmol/L) and 3-fold excess of complementary LNA-DNA or cDNA was done in the hybridization buffer [25 mmol/L Tris-HCl (pH 7.5) and 100 mmol/L NaCl] by heating the samples at 68°C for 10 minutes and slowly cooling to room temperature. The samples were diluted 15 times, and 1 µL of each sample was incubated in a total volume of 10 µL in RNase H buffer [75 mmol/L KCl, 50 mmol/L Tris-HCl, 3 mmol/L MgCl2, 10 mmol/L DTT (pH 8.3)] for 0.5, 5, 15, and 60 minutes at 37°C, in the presence of 0.5 unit Escherichia coli RNase H (BioLabs). The reaction was stopped by addition of EDTA (final concentration = 83 mmol/L).

One-base ladder was obtained by subjecting the RNA strand to a limited alkaline hydrolysis in 50 mmol/L NaHCO3/Na2CO3 (pH 9), 1 mmol/L EDTA, 0.25 mg/mL tRNA at 90°C for 7 minutes. RNase H digestions and limited alkaline hydrolysis products were separated on denaturing 20% PAGE (19:1 acrylamide:bisacrylamide, 8 mol/L urea) at 56°C. The gels were exposed to autoradiography.

Apoptotic Assay
We measured the caspase activity using Apo-ONE Homogeneous Caspase-3/7 Assay (Promega, Madison, WI). At 48 and 72 hours, the LNA-transfected KYO-1 and K562 cells were diluted in triplicate to a concentration of 5 x 104 cells (25 µL per well) into a 96-well plate. Then Apo-ONE caspase-3/7 reagent (substrate was diluted 1:100 with the supplied buffer) was added to the samples in a 1:1 ratio. The measure of fluorescence of each well was read at an excitation wavelength of 485 ± 20 nm and an emission of 530 ± 25 nm (Spectra Max Gemini XS, Molecular Devices Corp., Sunnyvale, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Design of LNA-DNA Gapmers against Oncogene BCR/ABL
Leukemic KYO-1 and K562 cells contain the Philadelphia chromosome carrying the BCR/ABL fusion gene. The mRNA transcribed from this hybrid gene is characterized either by the b2a2 or the b3a2 junction, and both types of transcripts are translated into a 210-kDa protein (p210Bcr/Abl; refs. 4, 5, 31). Recent studies have shown that 21-mer small interfering RNA (siRNA) site-directed against the b2a2 or b3a2 junctions specifically suppress the expression of the BCR/ABL oncogene (32–35). In the current work, we have designed LNA-DNA chimeric oligonucleotides to target the b2a2 and b3a2 mRNA junctions in CML cells (b2a2 target: nucleotides 284–314, accession no. AJ131467; b3a2 target: nucleotides 361–380, accession no. AJ131466; Fig. 1). It is known that an antisense molecule may act through two mechanisms: (a) activation of RNase H that recognizes and degrades RNA:DNA hybrid duplexes (36); (b) arrest of translation at the ribosome (37). Previous studies have shown that oligonucleotides containing LNA modifications at the 3' and 5' ends are able to recruit RNase H and promote the degradation of the target RNA (26, 27). In light of these results, we designed 20/21 mer junction-specific LNA-DNA gapmers with a central DNA region of 13/14 nucleotides and 3' and 5' end blocks of 3/4 contiguous LNA monomers. As control molecules, we designed gapmers with the same base contents as the antisense molecules but with a different sequence. First, we asked whether the designed gapmers could be efficiently transferred in CML cells and where they might be localized. We incubated for 5 hours 1 µmol/L b3a2 LNA-DNA-F, labeled at the 5' terminus with fluorescein, with KYO1 and K562 cells, and observed the uptake by confocal microscopy and cell cytometry. The gapmer was delivered to CML cells either as a naked molecule (with and without electroporation) or with a carrier (Lipofectamine 2000). The results are reported in Fig. 2A to F : left, nuclei stained with propidium iodide; middle, green fluorescence emitted by the gapmer taken up by the cells; right, superimposed views. It can be seen that when the delivery was assisted by a lipid carrier or by electroporation (single pulse at 500 V, 25 µF, {infty} {Omega}), all the cells seemed green, which indicates that they had efficiently taken up the gapmer. The green fluorescence seemed mainly localized in the cytoplasm but with a significant presence also in the nucleoli. It is noteworthy that KYO-1 cells and, to a lower extent, K562 cells are able to take up b3a2 LNA-DNA-F also when the gapmer was delivered alone, without any transfection assistance (Fig. 2A and D). The cellular uptake was also analyzed by quantitative flow cytometry. Figure 2G shows the percentage of CML cells with a fluorescence above the background level (untreated cells). In keeping with the confocal microscopy, the data show that KYO-1 cells assume efficiently the gapmer, even by simple diffusion, whereas in K562, the uptake seemed effective only in the presence of Lipofectamine or electroporation. Based on these results, to evaluate the biological effects, we decided to deliver the designed gapmers to CML cells by electroporation.


Figure 2
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  		Figure 2. A to F, confocal images of KYO-1 and K562 cells treated with 1 µmol/L b2a2 LNA-DNA-F, which was delivered to the cells either by electroporation or complexed with Lipofectamine 2000. Left, nuclei stained with propidium iodide; middle, green light emitted by the fluorescein-labeled gapmer; right, superimposed images. G, quantification of uptake in KYO-1 and K562 cells treated with 1 µmol/L b3a2 LNA-DNA-F. Ordinate, % cells with fluorescence above the background (untreated cells). Column 1, cells treated with the gapmer; column 2, cells treated with the gapmer and electroporated; column 3, cells treated with the gapmer complexed with Lipofectamine 2000.

 
To determine the affinity of the designed LNA-DNA gapmers for the b2a2 and b3a2 RNA junctions, we did circular dichroism experiments as a function of temperature. The circular dichroism spectra of the hybrid duplexes formed by b2a2 and b3a2 LNA-DNAs with the complementary RNA strands are dominated by a large and positive ellipticity at 265 nm and a weak and negative ellipticity at 240 nm (data not shown). The spectra indicate that the hybrid duplexes adopt a conformation close to that of A-DNA (38). Measuring the circular dichroism spectra at increasing temperatures, the 265-nm ellipticity decreased as the hybrid duplexes denatured. Plotting the circular dichroism signal at 264 nm as a function of temperature, we obtained two cooperative melting curves from which we evaluated a melting temperatures of 75°C for the b3a2 LNA-DNA:RNA and 79°C for b2a2 LNA-DNA:RNA. Considering that the melting curves have been obtained at a ionic strength of 20 mmol/L NaCl, the designed gapmers are expected to have, under cytoplasmic ionic strength conditions (~150 mmol/L KCl), an even higher affinity for their RNA targets (melting temperature > 80°C).

LNA-DNA Gapmers Induce RNase H Cleavage of BCR/ABL b2a2 and b3a2 RNA Targets
Antisense oligonucleotides can reduce the level of mRNA by activating RNase H, which hydrolyses RNA in RNA/DNA duplexes (39). We investigated the ability of the designed LNA-DNA gapmers to activate RNase H and promote the cleavage of BCR/ABL b2a2 and b3a2 mRNA junctions. Figure 3A shows the cleavage pattern obtained with b2a2 RNA duplexed with a complementary oligodeoxynucleotide or with b2a2 LNA-DNA gapmer. The resulting hybrid duplexes were exposed to RNase H up to 60 minutes. For convenience, we used RNase H from E. coli, as the catalytic properties of this enzyme are substantially similar to those of the mammalian enzyme (40). In the DNA/RNA heteroduplex, cleavage occurs at two main sites within few minutes. Comparison with a one-base ladder, obtained by a limited alkaline hydrolysis of the heteroduplex RNA sequence, showed that cleavage occurred at A10-G11 and A6-A7. When the DNA strand of the heteroduplex is replaced with b2a2 LNA-DNA, the main cleavage site is observed between A10 and G11 (i.e., 10 nucleotides from the 5'-RNA/3'-DNA terminus of the hybrid duplex). Figure 3B shows the cleavage pattern obtained with b3a2 RNA duplexed with DNA or b3a2 LNA-DNA gapmer. In this case, the DNA:RNA heteroduplex shows cleavage sites at A13-A14, A7-G8, and G6-A7. The corresponding duplex formed by b3a2 LNA-DNA exhibits a cleavage pattern characterized by two main cleavage sites: A13-A14 and U10-C11. With this heteroduplex too, the preferred cleavage site (U10-C11) is located 10 nucleotides from the 5'-RNA/3'-DNA terminus of the hybrid duplex. In summary, these enzymatic assays show that the heteroduplexes formed by the designed LNA-DNA gapmers and the b2a2 and b3a2 RNA targets are able to recruit RNase H and efficiently induce the cleavage of the BCR/ABL transcripts.


Figure 3
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  		Figure 3. A, digestion of the b2a2 DNA/RNA and b2a2 LNA-DNA:RNA heteroduplexes with E. coli RNase H. Lane 1, one-base ladder obtained by limited alkaline hydrolysis of the RNA strand; lanes 2 to 5, cleavage products of 0, 0.5, 15, and 60 min of incubation of b2a2 DNA:RNA heteroduplex with 0.5 unit RNase H; lanes 6 to 9, cleavage products of 0, 0.5, 15, and 60 min of incubation of the b2a2 LNA-DNA:RNA heteroduplex with 0.5 unit RNase H. B, digestion of b3a2 DNA:RNA and b3a2 LNA-DNA:RNA heteroduplexes with 0.5 unit E. coli RNase H. Lanes 1 and 2, untreated heteroduplexes; lane 11, one-base ladder obtained by limited alkaline hydrolysis of the RNA strand; lanes 3 to 6, cleavage products of 0.5, 5, 15, and 60 min of incubation of the DNA:RNA heteroduplex with 0.5 unit RNase H; lanes 7 to 10, cleavage products of 0.5, 5, 15, and 60 min of incubation of the b3a2 LNA-DNA:RNA heteroduplex with 0.5 unit RNase H. The RNA strand in the heteroduplexes was labeled with [{gamma}-32P]ATP and T4 polynucleotide kinase.

 
Site-Directed Targeting of b2a2 and b3a2 RNA Junctions and Transcript Suppression
To investigate the capacity of the designed antisense gapmers to reduce the level of the b3a2 and b2a2 transcripts in K562 and KYO-1 cells, we first did competitive reverse transcription-PCR experiments. Figure 4 reports the results of a typical experiment showing the level of b2a2 BCR/ABL transcript in KYO-1 cells, 5 hours following electroporation. Total mRNA was extracted from an equal number (1 x 106) of oligonucleotide-treated KYO-1 (target) and untreated K562 cells (competitor). The extracted mRNAs were mixed together, retrotranscribed in cDNA, and amplified by PCR using the BCR/ABL primers (see Materials and Methods). As target and competitor share the same primer sequences, the amplification gives rise to two DNA fragments: one of 387 bp from untreated K562 cells, the other of 312 bp from the oligonucleotide-treated KYO-1 cells. The PCR products were separated by polyacrylamide electrophoresis, and the two bands were quantified. The ratio between b2a2 mRNA (312 bp) and b3a2 mRNA (387 bp) in treated (T) and untreated (C) cells provided an estimate of the relative amount of target b2a2 mRNA compared with control b3a2 mRNA. The results reported as histograms show that the level of the b2a2 mRNA in KYO-1 cells treated with 0.1, 0.5, and 1 µmol/L b2a2 LNA-DNA is reduced in a dose-dependent manner, whereas control b2a2 cLNA-DNA does not affect the transcript level (Fig. 4). It is noteworthy that 1 µmol/L antisense oligonucleotide drastically reduces the level of b2a2 mRNA (~20% of control). As a loading control, we measured the level of the ABL transcript in each sample. A very similar result was obtained when b2a2 LNA-DNA was delivered to KYO-1 cells without electroporation (and liposomes; data not shown); this correlates with the finding that the gapmer is capable to diffuse into KYO-1 cells. Twenty-four hours after electroporation, we found that the b2a2 transcript had recovered to the level as in untreated cells. We also quantified the b2a2 mRNA following an alternative approach. We amplified in the same KYO-1 mRNA extract the BCR/ABL and glyceraldehyde-3-phosphate dehydrogenase transcripts. The ratio between BCR/ABL and glyceraldehyde-3-phosphate dehydrogenase in treated (T) and untreated (C) cells allowed to estimate the BCR/ABL transcripts relatively to glyceraldehyde-3-phosphate dehydrogenase (Fig. 5A ). The results obtained were in nice agreement with those shown in Fig. 4. These methods were also employed to determine the level b3a2 mRNA in K562 cells (Fig. 5B). In these cells, 1 µmol/L b3a2 LNA-DNA promoted a weaker but still significant suppression of the b3a2 mRNA (~50% of control) 5 hours after electroporation. As observed with KYO-1 cells, 24 hours following electroporation, the b3a2 transcript had recovered to the normal level.


Figure 4
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  		Figure 4. A, schematic representation of the competitive reverse transcription-PCR done to quantify mRNA in KYO-1 cells. Untreated K562 cells were used as competitor. EA122, EA500, and ABL 1A are the primer used (see Materials and Methods). B, typical competitive reverse transcription-PCR (5 h following electroporation) showing that 0.1, 0.5, and 1 µmol/L b2a2 LNA-DNA gapmer suppressed the level of b2a2 mRNA in KYO-1 cells. As a loading control, a 128-bp fragment from the wild-type ABL gene was amplified. C, % residual b2a2 mRNA in KYO-1 cells, 5 h after electroporation. % Residual mRNA is given by (T/C) x 100, where C is the 312/387 bp ratio in untreated cells, whereas T is the same ratio in oligonucleotide-treated cells. The data obtained were then normalized to ABL. Column 1, untreated cells; columns 2 to 4, cells treated with 0.1, 0.5, and 1 µmol/L b2a2 gapmer; columns 5 to 7, cells treated with 0.1, 0.5, and 1 µmol/L control b2a2 cLNA-DNA gapmer. The reverse transcription-PCR experiments have been done two to three times, and the histogram bars have an uncertainty of about ±10%.

 

Figure 5
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  		Figure 5. A, typical reverse transcription-PCR (5 h following electroporation) showing the level of b2a2 mRNA relative to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in KYO-1 cells untreated and treated with antisense and control LNA-DNA gapmers. % Residual mRNA in KYO-1 cells, 5 h after electroporation. % Residual mRNA is given by (T/C) x 100 values, where C is the 312/334 bp ratio in untreated cells, whereas T is the same ratio in oligonucleotide-treated cells. Column 1, untreated cells; columns 2 to 4, cells treated with 0.1, 0.5, and 1 µmol/L b2a2 LNA-DNA gapmer; columns 5 to 7, cells treated with 0.1, 0.5, and 1 µmol/L control b2a2 cLNA-DNA gapmer. B, levels of b3a2 mRNA in K562 cells, 5 h after electroporation. Ordinate, % residual b3a2 mRNA. % Residual mRNA is given by (T/C) x 100 values, where C is the 387/334 bp ratio in untreated cells, whereas T is the same ratio in oligonucleotide-treated cells. Column 1, untreated cells; columns 2 to 4, cells treated with 0.1, 0.5, and 1 µmol/L b3a2 LNA-DNA gapmer; columns 5 to 7, cells treated with 0.1, 0.5, and 1 µmol/L control b3a2 cLNA-DNA gapmer. The reverse transcription-PCR experiments have been done two to three times, and the histogram bars have an uncertainty of about ±10%.

 
Effect of LNA-DNA Antisense Oligonucleotides on Bcr/Abl Protein Level
As protein Bcr/Abl (p210 Bcr/Abl) has a relatively long half-life (~40 hours; ref. 41), we measured its level in treated and untreated K562 and KYO-1 cells by immunoblotting, 48 and 72 hours following electroporation. If the antisense oligonucleotides once introduced in the cells completely inhibit the expression of BCR/ABL, the level of protein Bcr/Abl, 48 and 72 hours after electroporation, should be around 40% and 25% of control, respectively. Figure 6A shows a typical Western blot obtained with KYO-1 cells treated for 72 hours with 1 and 5 µmol/L b2a2 LNA-DNA and control oligonucleotide. The level of protein Bcr/Abl in KYO-1 cells treated with 1, 2.5, and 5 µmol/L b2a2 LNA-DNA is shown in a histogram. At each concentration used, antisense b2a2 LNA-DNA, but not control b2a2 cLNA-DNA, strongly reduces the level of protein Bcr/Abl, up to 30% of control: a finding that correlates with the observed b2a2 transcript suppression. Figure 6B shows a Western blot obtained with K562 cells. As observed in previous studies (32, 35), in which K562 cells were treated with siRNAs, the level of Bcr/Abl protein was found significantly suppressed 48 hours after treatment. It can be seen that b3a2 LNA-DNA, used in the concentration range of 1 to 5 µmol/L, reduced the protein level to 40% of control. It is noteworthy that total protein suppression was unlikely to obtain in both types of cells, due to the long half-life of the Bcr/Abl protein. Western blots could not be done at longer times (>72 hours) because the cell division (every 18–20 hours) would "dilute" the antisense effect.


Figure 6
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  		Figure 6. A, Western blot analysis showing 72 h following electroporation the levels of Bcr/Abl protein and ß-actin in KYO-1 cells treated with antisense and control LNA-DNA gapmers. % Residual Bcr/Abl protein. % Residual Bcr/Abl protein is given by (T/C) x 100 values, where T is the ratio between protein Bcr/Abl and ß-actin in KYO-1 cells treated with antisense b2a2 LNA-DNA, and C is the same ratio in KYO-1 cells treated with control b2a2 cLNA-DNA gapmer. Columns 1, 3, and 5, levels of Bcr/Abl protein, compared with control (columns 2,4, and 6), in KYO-1 cells treated with 1, 2.5, and 5 µmol/L b2a2 LNA-DNA. B, Western blot analysis 48 h following electroporation showing the levels of Bcr/Abl protein and ß-actin in K562 cells treated with antisense and control b3a2 LNA-DNA gapmers. Columns 1, 3, and 5, levels of Bcr/Abl protein, compared with control (columns 2, 4, and 6), in K562 cells treated with 1, 2.5, and 5 µmol/L b3a2 LNA-DNA.

 
Biological Effects Produced in Leukemia Cells by the LNA-DNA Antisense Oligonucleotides
After having shown that the designed LNA-DNA gapmers specifically silence the BCR/ABL oncogene, we determined whether they also influenced the proliferation of CML cells. The leukemogenic potential of protein Bcr/Abl resides in the fact that its uncontrolled kinase activity usurps the normal function of protein Abl (5). The cellular consequences due to the presence in CML cells of protein Bcr/Abl are a deregulated proliferation and a reduced apoptosis (1). Previous studies have shown that the inhibition of BCR/ABL results in a dose-dependent reduction of cell viability (32, 35, 42). Figure 7A shows that b2a2 LNA-DNA has a significant effect on KYO-1 proliferation. After a single electroporation, cell growth was transiently reduced by b2a2 LNA-DNA to about 60% of control. This behavior parallels that previously observed with KYO-1 cells treated with siRNAs (35). We then asked whether the inhibition of cell growth was due to apoptosis. To this aim, we did a caspase-3/7 assay 48 and 72 hours following electroporation. The apoptosis was measured by the appearance of a fluorescent product generated by the caspases. Figure 7B shows that KYO-1 cells electroporated either with control oligonucleotide or with buffer only have a basal level of caspase activity, which is significantly lower than that observed with the antisense-treated cells, at both 48 and 72 hours after electroporation. Figure 8 shows the results of the proliferation and apoptosis assays done with K562 cells. As observed with KYO-1 cells, the cellular growth is inhibited by the treatment with b3a2 LNA-DNA, and the caspase activity significantly increased when the cells were treated with 5 µmol/L antisense oligonucleotide.


Figure 7
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  		Figure 7. A, cell growth of KYO-1 cells untreated and treated with 1 or 5 µmol/L antisense or control LNA-DNA gapmers. Ordinate, % viable cells with respect to control. % Viable cells is given by (T/C) x 100, where T is the number of live cells (counted by trypan blue) in the presence of a gapmer, and C is the number of live cells in the absence of a gapmer. Column 1, untreated cells; columns 2 and 3, cells treated with 1 and 5 µmol/L b2a2 LNA-DNA; columns 4 and 5, cells treated with 1 and 5 µmol/L b2a2 cLNA-DNA. B, apoptosis assays at 48 and 72 h following electroporation with antisense and control oligonucleotides. Ordinate, % caspase-3/7 activity. Column 1, untreated cells; columns 2 and 4, cells treated with 1 and 5 µmol/L b2a2 LNA-DNA; columns 3 and 5, cells treated with 1 and 5 µmol/L b2a2 cLNA-DNA. *, P < 0.05, standard t test versus control.

 

Figure 8
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  		Figure 8. A, cell growth K562 cells untreated and treated with 1 or 5 µmol/L antisense or control LNA-containing oligonucleotides. Ordinate, % viable cells compared with control. % Viable cells is given by (T/C) x 100, where T is the number of live cells (counted by trypan blue) in the presence of gapmers, and C is the number of live cells in the absence of gapmers. Column 1, untreated cells; columns 2 and 3, cells treated with 1 and 5 µmol/L b3a2 LNA-DNA; columns 4 and 5, cells treated with 1 and 5 µmol/L b3a2 cLNA-DNA. B, ordinate, % caspase-3/7 activity. Column 1, untreated cells; columns 2 and 4, cells treated with 1 and 5 µmol/L b2a2 LNA-DNA; columns 3 and 5, cells treated with 1 and 5 µmol/L b2a2 cLNA-DNA. *, P < 0.05, standard t test versus control.

 
Finally we determined whether leukemic cells treated with antisense LNA-DNA gapmers are sensitized to treatment with STI571. This is a potent inhibitor of the tyrosine kinase activity of p210Bcr/Abl that has shown promise in clinic (43). A problem emerging with the use of STI571 is due to the fact that patients with acute Philadelphia-positive leukemia tend to develop resistance against this drug (44). In this case, a combined treatment with more chemotherapeutic agents would be necessary to eradicate the disease. We observed that whereas the b3a2-junction K562 cells showed a high sensitivity to STI571 (IC50 = 0.1 µmol/L), the b2a2-junction KYO-1 cells showed a significant resistance against this drug (IC50 = 0.9 µmol/L). We, therefore, did chemosensitization experiments with KYO-1 cells. The cells were first treated with 1 µmol/L b2a2 gapmer and after 8 hours with 0.25, 0.5, and 1 µmol/L STI571. Cell survival was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay 72 hours following the STI571 treatment. The results showed that the combined treatment (1 µmol/L antisense + 1 µmol/L STI571 or 1 µmol/L antisense + 0.5 µmol/L STI571) resulted in an additive antiproliferative effect (Fig. 9 ). In the presence of 1 µmol/L b2a2-gapmer, the IC50 for STI571 was reduced from 0.9 to 0.5 µmol/L.


Figure 9
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  		Figure 9. A, cell viability measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays after 48 h ({circ} and {triangleup}) and 72 h (bullet and {blacktriangleup}) of incubation with increasing amounts of STI571. B to D, KYO-1 cell viability measured at 72 h following treatments with b2a2 LNA-DNA and STI571.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we tested BCR/ABL antisense LNA-DNA gapmers as potential anticancer therapeutic agents for the treatment of leukemia. We have designed two LNA-modified antisense oligonucleotides complementary to the b2a2 and b3a2 mRNAs expressed in Philadelphia-positive cells. These oligonucleotides have a central region of 13/14 deoxyribonucleotides and 5' and 3' end blocks of 3 or 4 LNA monomers. Previous studies have shown that LNA-containing oligonucleotides form very stable duplexes when they bind to cognate RNA, with melting temperature increased up to 4°C to 5°C per LNA modification (26). Our designed LNA-DNA gapmers having seven LNA monomers exhibit a thermal stability in 20 mmol/L NaCl falling between 75°C and 79°C. Under physiologic ionic strength (150 mmol/L monovalent cations), the melting temperatures are expected to further increase by 4°C to 5°C (45). Thus, the designed gapmers have a high affinity for cognate RNA, and this is an essential prerequisite that an ideal antisense molecule must possess to be biologically active. The silencing of a gene by an antisense drug may have two mechanisms of action: (a) degradation of mRNA by RNase H (36) and (b) steric block of translation at the ribosome (37). We found that b2a2 LNA-DNA and to a minor extent b3a2 LNA-DNA reduced the level of the target mRNA in KYO-1 and K562 cells. This suggests that the designed antisense gapmers are able to recruit RNase H and induce the cleavage of the transcripts. This is in perfect agreement with the finding that in a cell-free system, the hybrid duplexes formed by the designed LNA-DNA gapmers, and the target b2a2 and b3a2 RNAs are recognized by E. coli RNase H and cleaved. The cleavage sites occur in the RNA strand about 10 nucleotides from the 5'-RNA/3'-DNA terminus of the hybrid duplex, preferentially at AG and UC dinucleotides. Although the enzymatic properties of E. coli RNase H are not very different from those of the mammalian enzyme (40), the latter displays a cleavage preference for GU and GG dinucleotides (46). Considering that only b2a2 mRNA contains an optimal cleavage site (GG), it is possible that the mammalian RNase H enzyme degrades more efficiently the b2a2 RNA than the b3a2 RNA. This could explain why the level of b3a2 mRNA in the treated K562 cells is higher than that observed in treated KYO-1 cells.

An important property that LNA-containing oligonucleotides show is their higher resistance to nucleases compared with unmodified DNA. The half-lives of LNA-DNA gapmers in human or rat serum vary from 6 hours (27) to 14 to 16 hours (26). The fact that a reduced level of the BCR-ABL transcript was observed only at 5 hours after electroporation and not at 24 hours may be due to (a) a partial oligonucleotide degradation occurring under intracellular conditions after 14 to 16 hours and (b) a cell division occurring every 18 to 20 hours.

The fact that LNA structurally mimics the C3-endo sugar conformation that characterizes RNA (47), one wonders whether the designed gapmers activate a RNA interference–mediated silencing mechanism of the BCR/ABL oncogene. In fact, the designed LNA-DNA gapmers suppress the expression of BCR/ABL as efficiently as siRNA does (32–35). Three observations argue against this hypothesis. First, in this work, the reduction of b2a2 mRNA in KYO-1 cells was observed 5 hours after electroporation, but with siRNA, it was detected 24 hours following electroporation (32). This difference in response time is likely to reflect a different mechanism of action. In fact, whereas the RISC-mediated cleavage of mRNA promoted by siRNA is a complex process involving several proteins, the recruitment of RNase H by the antisense LNA-DNA gapmers is relatively fast. Under in vitro conditions, RNase H was found to cleave the b2a2 and b3a2 duplexes within 1 hour. Second, the silencing of BCR/ABL by the designed LNA-DNA gapmers are clearly observed at 1 µmol/L (i.e., at a concentration that is at least 10-fold higher than that used with siRNA). Third, it has been shown that the introduction of LNA modifications at the 5' terminus of a siRNA oligonucleotide substantially impaired the silencing activity by the RISC mechanism (48).

The potency of LNA-containing oligonucleotides as antisense molecules has been observed by different research groups, but most used as target a reporter gene (25, 49–52). In vivo, the activity of LNA-DNA gapmers has been first shown by Wahlestedt et al. (28), who injected the antisense oligonucleotides in the cerebrospinal fluid of rats to inhibit an antinociceptive response. Fluiter et al. (27) showed that LNA oligonucleotides targeted to the gene of the large subunit of RNA polymerase II strongly reduced the protein level and tumor growth. The same authors in a subsequent study used LNA-DNA gapmers to suppress specifically H-RAS mRNA and to reduce tumor growth in nude mice with tumor xenograft (53).

In our study, we have targeted a unique sequence present in the Philadelphia chromosome of CML cells. The designed b2a2 and b3a2 LNA-DNA gapmers showed a strong capacity to knockdown both the transcript and protein levels. The potency of the designed gapmers to silence the chimeric BCL/ABL gene is comparable with that obtained with siRNA, in terms of transcript and protein suppression, reduced proliferation, and activation of apoptosis (32). However, the use of siRNA in vivo and their possible use in therapeutics is still limited by a poor biostability in biological fluids and off-target effects. Conversely, LNA-containing oligonucleotides, being highly stable against nucleases and having a high affinity for RNA, seem to possess the features to become potent anticancer agents.

As a final consideration, we wish to point out that LNA-DNA gapmers may be used in combination with STI571. This inhibitor inactivates the tyrosine kinase activity of the Bcr/Abl oncoprotein and, since 1998, is used in the treatment of leukemia (43, 44). However, the emergence of resistance to STI571 has been recently recognized as a major problem in the treatment of Philadelphia-positive leukemia (44). In vitro studies suggested that the resistance may be due to BCR/ABL amplification or point mutations in the kinase domain of the protein (44). As LNA-DNA gapmers are nuclease resistant and able to strongly reduce the level of p210Bcr/Abl, their use in combination with STI571 may reduce resistance. We found that STI571 exhibited enhanced antiproliferative activity in the presence of 1 µmol/L antisense b2a2-specific gapmer. The IC50 of STI571, measured in KYO-1 cells exposed to the chemotherapeutics for 72 hours, was reduced from 0.9 to 0.5 µmol/L. This additive effect can probably be enhanced by increasing the concentrations of both the antisense oligonucleotide and STI571.

In conclusion, the present study shows that antisense LNA-DNA gapmers are powerful molecules to down-regulate BCR/ABL in Philadelphia-positive leukemic cells. When they are used in combination with STI571, an enhanced antiproliferative effect on refractory KYO-1 cells is observed. Although in vitro studies are not always predictive of clinical activities, our results show that LNA-DNA gapmers alone or in combination with STI571, may be used for the treatment of leukemia and for the purging of Philadelphia-positive cells.


   Acknowledgments
 
We thank Dr. Marina Giunta (Polyclinic of Udine) for giving advice in carrying out experiments with STI571.


   Footnotes
 
Grant support: Ministry of Education (PRIN 2005).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 1/ 4/06; revised 5/11/06; accepted 5/16/06.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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