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Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture
Cancer Letters 152 (2000) 135±143
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Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture M. Helena Vasconcelos, Sandra S. Beleza, Catriona Quirk, LuõÂs F. Maia, Clara Sambade, Jose E. GuimaraÄes* Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr. Roberto Frias, s/n, 4200 Porto, Portugal Received 29 September 1999; received in revised form 11 December 1999; accepted 11 December 1999
Abstract The synergistic use of antisense oligonucleotides (ASOs) towards the bcr-abl and the transferrin receptor (TfR) mRNA was studied in a chronic myeloid leukemia (CML) cell line, aiming to improve the ef®ciency of individual ASO treatment. At 20 mM concentration, bcr-abl ASOs reduced cell growth by 40% and was speci®c for cells that have the translocation: there was a 34% reduction of BCR-ABL protein. The TfR ASO reduced cell growth by 20% and decreased TfR protein by 24%. The ASOs were more potent at reducing cell growth when used in combination (respectively, 220 and 217% than bcr-abl ASO and TfR ASO when used individually at the 10 mM concentration), thus we postulate that there is synergism of action. Cell cycle analysis also revealed that the sub-G1 peak was bigger in the synergistic treatment. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antisense oligonucleotide; Leukemia; bcr-abl; Transferrin receptor
1. Introduction Antisense oligonucleotide technology has the potential to be used as therapy for several cancers with altered gene expression. The advantage of this technique is the selectivity of the approach, which relies on the hybridization of a short synthetic DNA strand, the antisense oligonucleotide (ASO), with the complementary mRNA sequence of the target gene. Chronic myeloid leukemia (CML) is an optimal target for antisense treatment since it is classically associated with a chromosomal translocation t(9;22), generating the bcr-abl fusion gene which codes for a tyrosine kinase (TK). It is documented that one of the * Corresponding author. Tel.: 1351-22-557-0700; fax: 135122-557-0799. E-mail address: [email protected] (J.E. GuimaraÄes)
activities of the BCR-ABL TK is to increase proliferation. Indeed, selective inhibition of leukemia cell proliferation has been achieved by antisense oligonucleotides directed towards the bcr-abl chimeric gene [1,2]. Furthermore, the BCR-ABL TK is a good target for antisense therapy because it has also been implicated in inhibition of apoptosis [3,4]. Others have attempted to approach leukemia therapy with ASOs designed towards the c-myb [5,6] or c-myc [7±9] oncogenes and the bcl-2 gene [10,11]. Despite the promising results obtained in cells in culture or even in animal studies [2], the ef®ciency of the technology needs to be enhanced without increasing toxicity. In this study we exploited the possibility of targeting simultaneously two genes involved in CML cell growth, the bcr-abl and the transferrin receptor (TfR) mRNAs. The advantage of using a
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00441-3
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combination of 2 ASOs is two-fold: ®rstly, the cells that `escape' the effect of one ASO may be destroyed by the other; secondly, simultaneous inhibition of two target genes may potentiate each other causing a synergistic inhibition of cell proliferation [12]. Others have used a similar approach, targeting simultaneously the bcr-abl and the c-myc mRNAs [13±15], having shown a synergistic effect on inhibition of proliferation in vitro and prolongation of survival of leukemic mice tested with a combination of bcr-abl and c-myc ASOs. Even though c-myc is expressed ubiquitously, the use of c-myc ASO at non-toxic concentrations together with bcr-abl ASO, did not cause toxicity [14,15]. We selected TfR as one of the target mRNAs because TfR is overexpressed in malignant proliferating cells, since the cell cycle enzyme ribonucleotide reductase has a strong requirement for iron and TfR is the major gateway for uptake of iron into the cell. Although the normal hemopoietic cells also need iron for proliferation, it is known that most of the hemopoietic stem cells are in a quiescent state with putatively lower iron requirements. This would allow for a presumably selective effect of a TfR ASO towards malignant cells. Previous work carried out by some of us [16] revealed that an ASO designed towards the TfR mRNA inhibited the growth of BV173 cells by 36.3%. 2. Materials and methods 2.1. Oligonucleotides The TfR oligonucleotides were designed using the GenEMBL database and further analyzed with the FASTA program to con®rm that they did not crossreact with other known human mRNAs. The ®nal sequences were as follows: ASO: 5 0 -ATCTAGCTTGATCCATCAT-3 0 and SO: 5 0 -ATGATGGATCAAGCTAGAT-3 0 . The bcr-abl ASOs used were the ones designed by Szczylik et al. [1]. 2.2. Antisense treatment Phosphorothioates were purchased from Eurogentech (Belgium) already puri®ed. They were dissolved in sterile H2O, further diluted in RPMI 1640 to a ®nal concentration of 200 mM, ®lter sterilized (0.2 mM
®lter) and stored at 2808C until use. Oligonucleotides were studied in three human leukemic cell lines: BV173 (CML lymphoid), K562 (CML erythroblastic) and HL60 (promyelocytic). Except where indicated, cells were seeded in triplicate wells at a density of 5 £ 10 5/ml for BV173 or 2 £ 10 5/ml for K562 and HL60, in a 100 ml volume in 96-well tissue culture plates. Cells were cultured in RPMI supplemented with 10% FCS (heat inactivated at 658C for 30 min) and 2 mM l-glutamine and were incubated with 10 or 20 mM oligonucleotides, on day 1. When used in combination, bcr-abl and TfR ASO were added to the culture at the same time. Further additions of oligonucleotides were made (half the dose after 24 h and a fourth of the dose at 48 h). Cells were incubated for a period of 9 days in a humidi®ed incubator at 378C with 5% CO2 in air. 2.3. Cell growth and viability Cell growth and viability were assessed on days 4, 7 and 9 using the Trypan Blue exclusion assay in a BuÈrker hemocytometer. 2.4. Flow cytometric analysis of CD71 1 cells Cells (5 £ 10 5/ml) were treated as indicated in antisense treatment and assessed 48 h following treatment. At this time a cellular suspension in 50 ml PBS±1% FCS was incubated with 5 ml of FITC-conjugated anti-TfR (anti-CD71) antibody or FITC-conjugated IgG2a (Immune Source) for 10 min at room temperature, in the dark. Cells were then washed twice in 500 ml PBS±1% FCS and resuspended in 0.5 ml of 0.5% paraformaldehyde in PBS. Analysis of the labeled population was performed using a Epics XL-MCL Flow Cytometer (Coulter). 2.5. Northern blotting Cells were seeded at 10 £ 10 5/ml in 10 ml culture ¯asks and treated as indicated in antisense treatment. RNA was extracted at the following time points: 0, 24 and 48 h. For RNA extraction the method of Chomczynski and Sacchi was followed [17], using the Tripure Isolation Reagent (Boehringer Mannheim, Portugal). The amount of RNA was determined by reading absorbance at 260 nm. RNA species were separated by size in a denaturing 2.5 M formalde-
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hyde/0.8% agarose gel. RNA samples (15 mg) were run at 150 V for 3 h. RNA was transferred onto a nylon membrane Hybond N1 (Amersham, UK) by capillary blotting. RNA was ®xed to the membrane by baking for 2 h at 808C. Filters were probed with a 402 bp cDNA fragment for the b2a2 breakpoint region of the bcr-abl mRNA, obtained by PCR (see below). This probe was labeled by random labeling with the Megaprime DNA labeling system kit (Amersham) and [g- 32P]CTP (Satis, Portugal). Puri®cation of the probe was carried out with a Nick column (Pharmacia, Sweden). Filters were prehybridized overnight at 428C in 50% formamide, 5£ Denhardt's, 3£ SSC, 5 mM Na2HPO4, 5 mM NaH2PO4, 0.2% SDS, 200 mM salmon sperm DNA, 50 mM Hepes and 130 mM glycine. Hybridization was performed overnight in 10 ml of hybridization solution (9.6 ml of pre-hybridization solution, 0.2ml of 50£ Denhardt's and 0.2 ml of salmon sperm DNA) with 32P-labelled cDNA probe. After hybridization the ®lter was washed with 0.1£ SSC and 0.1% SDS for 30 min at 528C. Membranes were exposed to an X-ray ®lm (Kodak XOMAT, Sigma). 2.6. Preparation of probe for Northern blotting by reverse transcriptase and PCR for bcr-abl RNA extraction was performed as described above for the Northern blotting. Synthesis of cDNA was performed in a 20 ml volume in a solution containing 3 mg of RNA, 1.3 mg of random oligonucleotide (Pharmacia), 50 mM Tris±HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT (Gibco), 125 mM of dATP, dCTP, dGTP and dTTP (Pharmacia Biotech) and 10 000 units/ml of Moloney murine leukemia virus reverse transcriptase (Gibco). The reaction was performed in a Gene Amp PCR System model 2400 (Perkin±Elmer) at 428C for 50 min and then at 708C for 15 min. The resulting cDNA fragments were ampli®ed in a total volume reaction mixture of 20 ml consisting of the following: 1 ml of cDNA, 20 mM Tris±HCl (pH 8.4), 50 mM KCl, 250 mM of dATP, dCTP, dGTP and dTTP, 1.75mM MgCl2, 40 units/ml of Thermus aquaticus YT1 Taq DNA polymerase (Gibco BRL) and 0.25 mM of each (upstream and downstream) primer. Reaction was carried out in a Gene Amp PCR System model 2400 (Perkin±Elmer) at 958C for 30 s, 628C for 50 s and
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728C for 1 min, for 35 cycles, with a ®nal extension at 728C for 10 min. The PCR products were separated on a 2% agarose gel in TBE, run for 1 h at 90 V. The cDNA was extracted from the gel using the QIAEX II gel extraction kit (Qiagen), cDNA was quanti®ed measuring OD at 260 nm. 2.7. Western blotting Cells were seeded at 10 £ 10 5/ml in 10 ml culture ¯asks and treated as indicated in antisense treatment. Protein was extracted at the following time points: 0, 48, 72 and 96 h. Brie¯y, cells were centrifuged at 1500 rev./min for 5±10 min at 48C, washed once with PBS, then centrifuged again at 1500 rev./min for 5±10 min at 48C. Cells were lysed in Winman's buffer (1% NP-40, 0.1 M Tris±HCl (pH 8.0), 0.15 M NaCl and 5 mM EDTA) with EDTA-free protease inhibitor cocktail (Boehringer Mannheim), vortexed, agitated at 48C for 15±20min, centrifuged at 13 000 rev./min for 10 min at 48C and the supernatants were kept frozen at 2708C. Proteins were quanti®ed with the BCA protein assay kit (Pierce, Portugal). Protein samples were prepared for the Western blotting by incubating the following at 708C for 10 min: 20 mg of protein, 2.5 ml of NuPage LDS sample buffer (Novex, Germany), 1 ml of NuPage sample reductor agent (10£, Novex) and water to make the volume up to 10 ml. Samples were vortexed before loading the gel (3±8% Tris±acetate from Novex). The gel was run at 150 V for 1 h with Tris±acetate SDS running buffer (1£, Novex) with NuPage antioxidant agent (Novex). Samples were blotted into a nitrocellulose membrane (Satis) with the Novex Electrophoresis System and transfer buffer (10% methanol, 5% transfer buffer (Novex) and 1% antioxidant agent (Novex)). The membrane was blocked for 1 h at room temperature with agitation with 5% non-fat dried milk, 0.05% (v/v) T-TBS, washed in 0.05% T-TBS and incubated with 2.5 mg/ml of mouse anti-c-abl primary antibody (Santa Cruz Biotechnology, Germany) overnight at 48C with agitation. After thoroughly washing with wash buffer, the membrane was incubated with the second antibody anti-mouse IgG -HRP (1:2000) and the streptavidin-HRP (1:5000) in 0.05% T-TBS, for 1 h at room temperature with agitation. The membrane was thoroughly washed again and detection was performed with the ECL Amersham kit
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(Satis). Brie¯y, detection solutions A and B were mixed in a ratio of 1:1 and the membrane was let in contact with this solution for 1 min at room temperature. Films were obtained by exposing the Hyper®lm ECL (Satis) to the membranes and developed and ®xed with the Processing chemicals Kodak GBX developer and ®xer twin pack (Sigma, Spain). Membranes were reprobed for actin following the same protocol. The dilutions of the goat anti-actinHRP primary antibody (Santa Cruz Biotechnology) and the anti-goat IgG-HRP secondary antibody (Santa Cruz Biotechnology) were 1:100 and 1:1000, respectively.
on the effect on cell growth of the ASO to the SO, thus indicating that the ASO is speci®c for cells bearing the b2a2 translocation (Fig. 1b,c). In K562 and HL60, cytotoxicity of the oligonucleotides at the 20 mM concentration was pronounced since there was a reduction in the number of viable cells following treatment with either oligonucleotide (AS or S)
2.8. Cell cycle analysis Cells were treated as in antisense treatment. Samples were processed for cell cycle analysis at days 2 and 7 following treatment with 20 mM oligonucleotides, by ®xation with 70% ethanol and redilution in a mixture of propidium iodide and RNase. Cell cycle pro®le was determined by ¯ow cytometry (Epics XL-MCL, Coulter) analyzing at least 10 000 cells. 2.9. Statistical analysis Differences between ASO and SO treatments were analyzed using a paired t-test (StatView 4.02 for MacIntosh). 3. Results 3.1. Growth suppression of CML cell lines after individual treatment with ASOs In an attempt to determine if the bcr-abl ASO was speci®c for the b2a2 translocation for which it had been designed [1], 20 mM ASO or SO were added to triplicate cultures of BV173 (Fig. 1a), K562 (Fig. 1b) or HL60 (Fig. 1c) cells in culture on day 1. Half of the doses were added on day 2 and a fourth on day 3. In the BV173 cell line, with the b2a2 translocation, the ASO reduced viable cells by nearly 40% on day 9, as compared with the control oligonucleotide or with no treatment (Fig. 1a). In the K562 cell line (with a b3a2 translocation) and in the HL60 cell line (neither b2a2 nor b3a2 translocation) there was no difference
Fig. 1. Effect of bcr-abl ASO or SO in the viable cell number of different cell lines. BV173 cells (starting concentration 5 £ 10 5/ml) (a), K562 cells (2 £ 10 5/ml) (b) or HL60 cells (2 £ 10 5/ml) (c) were treated with 20 mM ASO or SO or with control media at day 1, 10 mM at 24 h and 5 mM at 48 h. Viable cells were counted at days 4, 7 and 9. Results are the mean of three independent experiments (three replicates each). * Represents P # 0:05 between AS and control treatments and 1 represents P # 0:05 between S and control treatments.
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when compared with control cells. In the BV173 cell line such cytotoxicity was not found at day 9. A study was performed on BV173 by comparing the response of cells treated as above with cells treated with half dose, that is 10 mM on day 1, 5 mM on day 2 and 2.5 mM on day 3. Table 1 represents the data obtained on day 7 of treatment. At 10 mM there was no cell death caused either by the sense or the antisense oligonucleotides. The effect of the TfR ASO was also tested in the BV173 cell line, under the same treatment conditions as the bcr-abl ASO. The ASO reduced the number of viable cells by nearly 30% on day 9, as compared with no treatment (Fig. 2). However, the SO also reduced viable cell number, at this concentration. In order to reduce this cell death, a study was performed at reduced oligo concentration as for the bcr-abl oligonucleotides. The TfR SO was less cytotoxic at 10 mM than at 20 mM (Table 1). However, as in the case of bcr-abl, the TfR ASO is less effective at 10 mM than at 20 mM, if compared with the SO treatment. 3.2. Growth suppression of CML cell lines following concomitant treatment with both ASOs A combined treatment with both oligonucleotides was also carried out, using a 20 mM concentration of Table 1 Number of viable cells obtained by individual or concomitant ASO treatment of BV173 for 7 days a Treatment
Number of % of SO ^ SE (n) viable cells (£10 5/ml) ^ SE (n)
Control (medium only) bcr-abl ASO bcr-abl SO TfR ASO TfR SO bcr-abl 1 TfR ASO bcr-abl 1 TfR SO
41.6 ^ 3.1 37.7 ^ 4.3 40.0 ^ 5.5 35.7 ^ 4.5 39.5 ^ 6.8 27.7 ^ 5.5 37.0 ^ 3.6
(3) (3) (3) (3) (3) (3) (3)
± 95 ^ 7 (3) ± 92 ^ 5 (3) ± 75 ^ 12 (3) ±
a Cultures of BV173 cells (5 £ 10 5/ml) were treated with bcr-abl or TfR or with a combination of both bcr-abl and TfR oligonucleotides. Treatments were with 10 mM of each oligonucleotide individually or with a combination of both oligonucleotides (5 mM of each). These concentrations of oligonucleotides were added at day 1 and were followed by treatment with half the dose at 24 h and a quarter of the dose at 48 h. Results are the mean of three independent experiments (three replicates each).
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Fig. 2. Effect of TfR ASO or SO in the viable cell number of BV173 cells. Cells (5 £ 10 5/ml) were treated with 20 mM ASO or SO or with control media at day 1, 10 mM at 24 h and 5 mM at 48 h. Viable cells were counted at days 4, 7 and 9. Results are the mean of three independent experiments (three replicates each). * Represents P # 0:05 between AS and control treatments.
each oligonucleotide (total of 40 mM oligonucleotides concentration). Data proved that this treatment was very toxic to the cells as cell death was very high (results not shown). Using half the concentration of each oligonucleotide (10 mM each therefore a total of 20 mM oligonucleotide concentration) there was still toxicity caused by the sense oligonucleotide treatments. At this concentration, the ASO treatment reduced cell number by 22% (P 0:03) when compared with SO treatment (results not shown). Reducing the dose of the oligonucleotide treatment even further to 5 mM each (total of 10 mM oligonucleotide concentration) there was no toxic effect caused by the sense treatment and the concomitant antisense treatment reduced cell number by 25% when compared with the concomitant SO treatment, as compared with 5% reduction obtained with the bcrabl ASO and 8% reduction obtained with the TfR ASO at 10 mM (Table 1) in relation to the respective SO controls. Thus, the 10 mM synergistic treatment was more ef®cient in reducing cell growth in the BV173 cell line than the individual ASO treatments. 3.3. Speci®city of action To determine whether the bcr-abl ASO was speci®c towards its own mRNA, BV173 cells were treated with 20 mM AS or S oligonucleotides for 24 or 48 h, at which time points total mRNA was extracted and transferred into a membrane which was further probed
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with a bcr-abl cDNA speci®c for the b2a2 translocation. To compensate for differences in loading the membrane was reprobed for the GAPDH mRNA. Results indicate that there was only a slight reduction (ASO compared to SO treatment) of the bcr-abl mRNA at 24 h (8% reduction) and 48 h (11% reduction) following oligonucleotide treatment (Fig. 3a). Further to this study, the BCR-ABL protein levels were determined by Western blotting (Fig. 3b) 48, 72 and 96 h following treatment with either ASO or
SO. These results show a reduction of BCR-ABL (34% at 48 h and 15% at 72 and 96 h) when comparing the ASO with the SO treatment. The speci®city of the TfR ASO was studied by ¯ow cytometric analysis of CD711 cells. BV173 cells were treated with 20 mM ASO or SO for 48 h, at which time the cells were processed for ¯ow cytometric analysis, labeling them with a TfR antibody. Results (Fig. 4) indicated that 20 mM TfR ASO were capable of reducing TfR expression 48 h following treatment, as compared with the SO, by 24%. However, the SO also seemed to reduced the expression of TfR, when compared with the control cells, by nearly 20%. This correlates well with the cytotoxic effect detected for the TfR SO at a later time (Fig. 2). 3.4. Analysis of the cell cycle Studies of the cell cycle distribution by ¯ow cytometry revealed that either the individual treatments with ASOs or the concomitant treatment with both oligonucleotides (20 mM) caused an accumulation of cells in S phase by day 7, preventing cells from progressing in the cell cycle (Fig. 5). Furthermore, it was possible to observe a sub-G1 peak, which was bigger in the concomitant treatment with the two ASOs than in the individual treatments, suggesting an increase in apoptosis by day 7.
Fig. 3. Speci®city of the bcr-abl antisense effect. Effect at the mRNA level (a): Northern Blot of BV173 cells (10 £ 10 5/ml) treated with bcr-abl ASO or SO or with medium (no oligos). RNA was extracted at 24 h after treatment with 20 mM oligonucleotide or at 48 h after treatment with 20 mM oligonucleotide at day 1 and 10 mM at 24 h. The membrane was probed with a cDNA obtained by RTPCR and reprobed with GAPDH cDNA. Effect at the protein level (b): Western Blot of BV173 cells (10 £ 10 5/ml) treated with bcr-abl ASO or SO or with medium alone. Proteins were extracted at 48, 72 and 96 h after treatment with 20 mM oligonucleotide, 10 mM at 24 h and 5 mM at 48 h. The membrane was probed with an anti-c-abl primary antibody and reprobed with an actin antibody. Results shown in the text are comparison of bcr-abl/actin ratios in the different conditions.
Fig. 4. Speci®city of the TfR antisense effect. Flow cytometric analysis of TfR protein expression in BV173 cells 48 h after incubation with 20 mM oligonucleotide and 10 mM at 24 h.
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Fig. 5. Representative example of the effect of antisense treatment in the cell cycle pro®le. BV173 cells were treated with bcr-abl or TfR oligonucleotides or with both (with half the dose of each oligonucleotide). The total oligonucleotide added was 20 and 10 mM at 24 h and 5 mM at 48 h. Cell cycle pro®le was analyzed at days 2 and 7 after treatment.
4. Discussion Treatment with antisense oligonucleotides, on their own or combined with other cytotoxic agents, is a promising strategy in cancer therapy, mainly in stages of minimal disease. Diseases which have unique genes resulting from non-random chromosomal translocations are likely targets and this approach has been tested by basic and clinical researchers for the past few years. Amongst these fusion genes the most commonly tested has been the bcr-abl gene in CML [1,2,18]. However treatment with bcr-abl ASO has been proved so far insuf®cient to kill all malignant cells. In the present work, we have attempted to improve the speci®c killing capacity of ASO towards bcr-abl by concomitant treatment with ASO towards the TfR gene. Although TfR is ubiquitously expressed we
believe that a selective effect could be obtained due to a higher need of the malignant cells for iron. We designed a new ASO for TfR, which reduced BV173 viable cell number by nearly 30% at 20mM concentration, also decreasing TfR protein levels. The bcr-abl ASO used was the one designed by Szczylik et al. [1], which reduced BV173 viable cell number by 40% at 20 mM concentration in our hands. Similar results had been obtained by others, using the same oligonucleotide and the same concentration [18]. We found evidence for a small reduction of bcr-abl mRNA and protein levels; furthermore, this bcr-abl ASO was speci®c for the b2a2 translocation of the bcr-abl gene, as demonstrated by ASO treatment of control cell lines (K562 and HL60). This indicates that this ASO would be able to target the bcr-abl mRNA of malignant cells, without causing normal cell death.
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The TfR SO presented some toxicity to the cells at 20 mM but not at 10 mM concentration. In agreement to this, the less cytotoxic concomitant oligonucleotide treatment was the one carried out at 10 mM (total oligonucleotide concentration). Indeed, at this concentration the concomitant SO treatment did not cause cytotoxicity, whereas the concomitant ASO treatment reduced viable cell number by 25% when compared with the SO, 7 days after the beginning of the treatment. This reduction was greater than the one obtained by the individual use of the ASO (220 and 217% than bcr-abl ASO and TfR ASO alone) at 10mM concentration, thus proving the existence of a synergistic effect. When the non-speci®c toxicity of the oligonucleotides is reduced the antisense effect becomes more evident. The ef®cacy and speci®city of the oligonucleotides were also proved at a molecular level, by reducing the levels of the corresponding mRNAs or proteins. However, reduction in bcr-abl mRNA and protein levels was not as pronounced as one would expect for two possible reasons: (i) the ASO is not very ef®cient; (ii) the time points studied were not the ones at which levels of mRNA and protein are the lowest. Furthermore, both the individual ASO treatments and the concomitant treatment prevented progression in the cell cycle and increased the sub-G1 peak suggestive of apoptosis. This was more pronounced in the concomitant treatment with the two ASOs than in the individual treatments. Thus, we conclude that this approach might be useful in controlling proliferation of CML cells since even though we did not obtain a very strong reduction in viable cell number, all the results indicate that the bcr-abl ASO is speci®c to bcr-abl mRNA and that there is a synergism of action of both ASOs when used concomitantly. Better reduction of viable cell number would be expected if less toxic and more speci®c and potent oligonucleotides could be designed. In recent years several chemical modi®cations of oligonucleotides that improve uptake and potency without increasing toxicity have been designed (reviewed in [12]). Alternatively, liposomes [19,20] or Streptolysin O [21,22] may be used to improve cellular uptake of oligonucleotides, therefore increasing ef®ciency. We are currently investigating these possibilities of improving antisense oligonucleotide ef®cacy.
Acknowledgements We thank Professor C. Szczylik for proof-reading this manuscript and Dr Filipe Sansonetty's team (IPATIMUP) for analysis on the Flow Cytometer. This work was supported by FCT (Portugal), project PRAXIS/2/2.1/SAU/1283/95. Sandra Beleza has a BIC grant and M. Helena Vasconcelos has a postdoctoral grant from FCT. References [1] C. Szczylik, T. Skorski, N.C. Nicolaides, L. Manzella, L. Malaguarnera, D. Venturelli, A.M. Gewirtz, B. Calabretta, Selective inhibition of leukemia cell proliferation by BCRABL antisense oligodeoxynucleotides, Science 253 (1991) 562±565. [2] T. Skorski, M. Nieborowska-Skorska, N.C. Nicolaides, C. Szczylik, P. Iversen, R.V. Iozzo, G. Zon, B. Calabretta, Suppression of Philadelphia leukemia cell growth in mice by BCR-ABL antisense oligodeoxynucleotide, Proc. Natl. Acad. Sci. USA 91 (1994) 4504±4508. [3] T.F.C.M. Smetsers, T. Skorski, L.T.F. van de Locht, H.M.C. Wessels, A.H.M. Pennings, T. de Witte, B. Calabretta, E.J.B. Mensink, Antisense BCR-ABL oligonucleotides induce apoptosis in the Philadelphia chromosome-positive cell line BV173, Leukemia 8 (1) (1994) 129±140. [4] A. McGahon, R. Bissonnette, M. Schmitt, K.M. Cotter, D.R. Green, T.G. Cotter, BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death, Blood 83 (5) (1994) 1179±1187. [5] G. Anfossi, A.M. Gewirtz, B. Calabretta, An oligomer complementary to c-myb-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines, Proc. Natl. Acad. Sci. USA 86 (1989) 3379±3383. [6] B. Calabretta, R.B. Sims, M. Valtieri, D. Caracciolo, C. Szczylik, D. Venturelli, M. Ratajczak, M. Beran, A.M. Gewirtz, Normal and leukemic hematopoietic cells manifest differential sensitivity to inhibitory effects of c-myb antisense oligodeoxynucleotides: an in vitro study relevant to bone marrow purging, Proc. Natl. Acad. Sci. USA 88 (1991) 2351±2355. [7] R. Heikkila, G. Schwab, E. Wickstrom, S.L. Loke, D.H. Pluznik, R. Watt, L.M. Neckers, c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1, Nature 328 (1987) 445±449. [8] G. Degols, J.-P. Leonetti, N. Mechti, B. Lebleu, Antiproliferative effects of antisense oligonucleotides directed to the RNA of c-myc oncogene, Nucleic Acids Res. 19 (4) (1991) 945± 948. [9] T. Skorski, D. Perrotti, M. Nieborowska-Skorska, S. Gryaznov, B. Calabretta, Antileukemia effect of c-myc N3 0 -P5 0 phosphoramidate antisense oligonucleotides in vivo, Proc. Natl. Acad. Sci. USA 94 (1997) 3966±3971.
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Moreira, J.P. Ramos, An antisense oligonucleotide to the transferrin receptor inhibits proliferation of leukemic cells in vitro, Blood 84 (Suppl. 1) (1994) 607a. P. Chomczynski, N. Sacchi, Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem. 162 (1987) 156±159. T.F.C.M. Smetsers, L.T.F. van de Locht, A.H.M. Pennings, H.M.C. Wessels, T.M. de Witte, E.J.B. Mensink, Phosphorothioate BCR-ABL antisense oligonucleotides induce cell death, but fail to reduce cellular Bcr-Abl protein levels, Leukemia 9 (1995) 118±130. S. Akhtar, R.L. Juliano, Liposome delivery of antisense oligonucleotides: adsorption and ef¯ux characteristics of phosphorothioate oligodeoxynucleotides, J. Control Rel. 22 (1992) 47± 56. R. Kronenwett, U. Steidl, M. Kirsch, G. Sczakiel, R. Haas, Oligodeoxyribonucleotide uptake in primary human hematopoietic cells is enhanced by cationic lipids and depends on the hematopoietic cell subset, Blood 91 (3) (1998) 852±862. D.G. Spiller, D.M. Tidd, Nuclear delivery of antisense oligodeoxynucleotides through reversible permeabilization of human leukemia cells with Streptolysin O, Antisense Res. Dev. 5 (1995) 13±21. C.M. Broughton, D.G. Spiller, N. Pender, M. Komorovskaya, J. Grzybowski, R.V. Giles, D.M. Tidd, R.E. Clark, Preclinical studies of streptolysin-O in enhancing antisense oligonucleotide uptake in harvests from chronic myeloid leukaemia patients, Leukemia 11 (1997) 1435±1441.
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Limited synergistic effect of antisense oligonucleotides against bcr-abl and transferrin receptor mRNA in leukemic cells in culture M. Helena Vasconcelos, Sandra S. Beleza, Catriona Quirk, LuõÂs F. Maia, Clara Sambade, Jose E. GuimaraÄes* Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Rua Dr. Roberto Frias, s/n, 4200 Porto, Portugal Received 29 September 1999; received in revised form 11 December 1999; accepted 11 December 1999
Abstract The synergistic use of antisense oligonucleotides (ASOs) towards the bcr-abl and the transferrin receptor (TfR) mRNA was studied in a chronic myeloid leukemia (CML) cell line, aiming to improve the ef®ciency of individual ASO treatment. At 20 mM concentration, bcr-abl ASOs reduced cell growth by 40% and was speci®c for cells that have the translocation: there was a 34% reduction of BCR-ABL protein. The TfR ASO reduced cell growth by 20% and decreased TfR protein by 24%. The ASOs were more potent at reducing cell growth when used in combination (respectively, 220 and 217% than bcr-abl ASO and TfR ASO when used individually at the 10 mM concentration), thus we postulate that there is synergism of action. Cell cycle analysis also revealed that the sub-G1 peak was bigger in the synergistic treatment. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antisense oligonucleotide; Leukemia; bcr-abl; Transferrin receptor
1. Introduction Antisense oligonucleotide technology has the potential to be used as therapy for several cancers with altered gene expression. The advantage of this technique is the selectivity of the approach, which relies on the hybridization of a short synthetic DNA strand, the antisense oligonucleotide (ASO), with the complementary mRNA sequence of the target gene. Chronic myeloid leukemia (CML) is an optimal target for antisense treatment since it is classically associated with a chromosomal translocation t(9;22), generating the bcr-abl fusion gene which codes for a tyrosine kinase (TK). It is documented that one of the * Corresponding author. Tel.: 1351-22-557-0700; fax: 135122-557-0799. E-mail address: [email protected] (J.E. GuimaraÄes)
activities of the BCR-ABL TK is to increase proliferation. Indeed, selective inhibition of leukemia cell proliferation has been achieved by antisense oligonucleotides directed towards the bcr-abl chimeric gene [1,2]. Furthermore, the BCR-ABL TK is a good target for antisense therapy because it has also been implicated in inhibition of apoptosis [3,4]. Others have attempted to approach leukemia therapy with ASOs designed towards the c-myb [5,6] or c-myc [7±9] oncogenes and the bcl-2 gene [10,11]. Despite the promising results obtained in cells in culture or even in animal studies [2], the ef®ciency of the technology needs to be enhanced without increasing toxicity. In this study we exploited the possibility of targeting simultaneously two genes involved in CML cell growth, the bcr-abl and the transferrin receptor (TfR) mRNAs. The advantage of using a
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00441-3
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combination of 2 ASOs is two-fold: ®rstly, the cells that `escape' the effect of one ASO may be destroyed by the other; secondly, simultaneous inhibition of two target genes may potentiate each other causing a synergistic inhibition of cell proliferation [12]. Others have used a similar approach, targeting simultaneously the bcr-abl and the c-myc mRNAs [13±15], having shown a synergistic effect on inhibition of proliferation in vitro and prolongation of survival of leukemic mice tested with a combination of bcr-abl and c-myc ASOs. Even though c-myc is expressed ubiquitously, the use of c-myc ASO at non-toxic concentrations together with bcr-abl ASO, did not cause toxicity [14,15]. We selected TfR as one of the target mRNAs because TfR is overexpressed in malignant proliferating cells, since the cell cycle enzyme ribonucleotide reductase has a strong requirement for iron and TfR is the major gateway for uptake of iron into the cell. Although the normal hemopoietic cells also need iron for proliferation, it is known that most of the hemopoietic stem cells are in a quiescent state with putatively lower iron requirements. This would allow for a presumably selective effect of a TfR ASO towards malignant cells. Previous work carried out by some of us [16] revealed that an ASO designed towards the TfR mRNA inhibited the growth of BV173 cells by 36.3%. 2. Materials and methods 2.1. Oligonucleotides The TfR oligonucleotides were designed using the GenEMBL database and further analyzed with the FASTA program to con®rm that they did not crossreact with other known human mRNAs. The ®nal sequences were as follows: ASO: 5 0 -ATCTAGCTTGATCCATCAT-3 0 and SO: 5 0 -ATGATGGATCAAGCTAGAT-3 0 . The bcr-abl ASOs used were the ones designed by Szczylik et al. [1]. 2.2. Antisense treatment Phosphorothioates were purchased from Eurogentech (Belgium) already puri®ed. They were dissolved in sterile H2O, further diluted in RPMI 1640 to a ®nal concentration of 200 mM, ®lter sterilized (0.2 mM
®lter) and stored at 2808C until use. Oligonucleotides were studied in three human leukemic cell lines: BV173 (CML lymphoid), K562 (CML erythroblastic) and HL60 (promyelocytic). Except where indicated, cells were seeded in triplicate wells at a density of 5 £ 10 5/ml for BV173 or 2 £ 10 5/ml for K562 and HL60, in a 100 ml volume in 96-well tissue culture plates. Cells were cultured in RPMI supplemented with 10% FCS (heat inactivated at 658C for 30 min) and 2 mM l-glutamine and were incubated with 10 or 20 mM oligonucleotides, on day 1. When used in combination, bcr-abl and TfR ASO were added to the culture at the same time. Further additions of oligonucleotides were made (half the dose after 24 h and a fourth of the dose at 48 h). Cells were incubated for a period of 9 days in a humidi®ed incubator at 378C with 5% CO2 in air. 2.3. Cell growth and viability Cell growth and viability were assessed on days 4, 7 and 9 using the Trypan Blue exclusion assay in a BuÈrker hemocytometer. 2.4. Flow cytometric analysis of CD71 1 cells Cells (5 £ 10 5/ml) were treated as indicated in antisense treatment and assessed 48 h following treatment. At this time a cellular suspension in 50 ml PBS±1% FCS was incubated with 5 ml of FITC-conjugated anti-TfR (anti-CD71) antibody or FITC-conjugated IgG2a (Immune Source) for 10 min at room temperature, in the dark. Cells were then washed twice in 500 ml PBS±1% FCS and resuspended in 0.5 ml of 0.5% paraformaldehyde in PBS. Analysis of the labeled population was performed using a Epics XL-MCL Flow Cytometer (Coulter). 2.5. Northern blotting Cells were seeded at 10 £ 10 5/ml in 10 ml culture ¯asks and treated as indicated in antisense treatment. RNA was extracted at the following time points: 0, 24 and 48 h. For RNA extraction the method of Chomczynski and Sacchi was followed [17], using the Tripure Isolation Reagent (Boehringer Mannheim, Portugal). The amount of RNA was determined by reading absorbance at 260 nm. RNA species were separated by size in a denaturing 2.5 M formalde-
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hyde/0.8% agarose gel. RNA samples (15 mg) were run at 150 V for 3 h. RNA was transferred onto a nylon membrane Hybond N1 (Amersham, UK) by capillary blotting. RNA was ®xed to the membrane by baking for 2 h at 808C. Filters were probed with a 402 bp cDNA fragment for the b2a2 breakpoint region of the bcr-abl mRNA, obtained by PCR (see below). This probe was labeled by random labeling with the Megaprime DNA labeling system kit (Amersham) and [g- 32P]CTP (Satis, Portugal). Puri®cation of the probe was carried out with a Nick column (Pharmacia, Sweden). Filters were prehybridized overnight at 428C in 50% formamide, 5£ Denhardt's, 3£ SSC, 5 mM Na2HPO4, 5 mM NaH2PO4, 0.2% SDS, 200 mM salmon sperm DNA, 50 mM Hepes and 130 mM glycine. Hybridization was performed overnight in 10 ml of hybridization solution (9.6 ml of pre-hybridization solution, 0.2ml of 50£ Denhardt's and 0.2 ml of salmon sperm DNA) with 32P-labelled cDNA probe. After hybridization the ®lter was washed with 0.1£ SSC and 0.1% SDS for 30 min at 528C. Membranes were exposed to an X-ray ®lm (Kodak XOMAT, Sigma). 2.6. Preparation of probe for Northern blotting by reverse transcriptase and PCR for bcr-abl RNA extraction was performed as described above for the Northern blotting. Synthesis of cDNA was performed in a 20 ml volume in a solution containing 3 mg of RNA, 1.3 mg of random oligonucleotide (Pharmacia), 50 mM Tris±HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT (Gibco), 125 mM of dATP, dCTP, dGTP and dTTP (Pharmacia Biotech) and 10 000 units/ml of Moloney murine leukemia virus reverse transcriptase (Gibco). The reaction was performed in a Gene Amp PCR System model 2400 (Perkin±Elmer) at 428C for 50 min and then at 708C for 15 min. The resulting cDNA fragments were ampli®ed in a total volume reaction mixture of 20 ml consisting of the following: 1 ml of cDNA, 20 mM Tris±HCl (pH 8.4), 50 mM KCl, 250 mM of dATP, dCTP, dGTP and dTTP, 1.75mM MgCl2, 40 units/ml of Thermus aquaticus YT1 Taq DNA polymerase (Gibco BRL) and 0.25 mM of each (upstream and downstream) primer. Reaction was carried out in a Gene Amp PCR System model 2400 (Perkin±Elmer) at 958C for 30 s, 628C for 50 s and
137
728C for 1 min, for 35 cycles, with a ®nal extension at 728C for 10 min. The PCR products were separated on a 2% agarose gel in TBE, run for 1 h at 90 V. The cDNA was extracted from the gel using the QIAEX II gel extraction kit (Qiagen), cDNA was quanti®ed measuring OD at 260 nm. 2.7. Western blotting Cells were seeded at 10 £ 10 5/ml in 10 ml culture ¯asks and treated as indicated in antisense treatment. Protein was extracted at the following time points: 0, 48, 72 and 96 h. Brie¯y, cells were centrifuged at 1500 rev./min for 5±10 min at 48C, washed once with PBS, then centrifuged again at 1500 rev./min for 5±10 min at 48C. Cells were lysed in Winman's buffer (1% NP-40, 0.1 M Tris±HCl (pH 8.0), 0.15 M NaCl and 5 mM EDTA) with EDTA-free protease inhibitor cocktail (Boehringer Mannheim), vortexed, agitated at 48C for 15±20min, centrifuged at 13 000 rev./min for 10 min at 48C and the supernatants were kept frozen at 2708C. Proteins were quanti®ed with the BCA protein assay kit (Pierce, Portugal). Protein samples were prepared for the Western blotting by incubating the following at 708C for 10 min: 20 mg of protein, 2.5 ml of NuPage LDS sample buffer (Novex, Germany), 1 ml of NuPage sample reductor agent (10£, Novex) and water to make the volume up to 10 ml. Samples were vortexed before loading the gel (3±8% Tris±acetate from Novex). The gel was run at 150 V for 1 h with Tris±acetate SDS running buffer (1£, Novex) with NuPage antioxidant agent (Novex). Samples were blotted into a nitrocellulose membrane (Satis) with the Novex Electrophoresis System and transfer buffer (10% methanol, 5% transfer buffer (Novex) and 1% antioxidant agent (Novex)). The membrane was blocked for 1 h at room temperature with agitation with 5% non-fat dried milk, 0.05% (v/v) T-TBS, washed in 0.05% T-TBS and incubated with 2.5 mg/ml of mouse anti-c-abl primary antibody (Santa Cruz Biotechnology, Germany) overnight at 48C with agitation. After thoroughly washing with wash buffer, the membrane was incubated with the second antibody anti-mouse IgG -HRP (1:2000) and the streptavidin-HRP (1:5000) in 0.05% T-TBS, for 1 h at room temperature with agitation. The membrane was thoroughly washed again and detection was performed with the ECL Amersham kit
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(Satis). Brie¯y, detection solutions A and B were mixed in a ratio of 1:1 and the membrane was let in contact with this solution for 1 min at room temperature. Films were obtained by exposing the Hyper®lm ECL (Satis) to the membranes and developed and ®xed with the Processing chemicals Kodak GBX developer and ®xer twin pack (Sigma, Spain). Membranes were reprobed for actin following the same protocol. The dilutions of the goat anti-actinHRP primary antibody (Santa Cruz Biotechnology) and the anti-goat IgG-HRP secondary antibody (Santa Cruz Biotechnology) were 1:100 and 1:1000, respectively.
on the effect on cell growth of the ASO to the SO, thus indicating that the ASO is speci®c for cells bearing the b2a2 translocation (Fig. 1b,c). In K562 and HL60, cytotoxicity of the oligonucleotides at the 20 mM concentration was pronounced since there was a reduction in the number of viable cells following treatment with either oligonucleotide (AS or S)
2.8. Cell cycle analysis Cells were treated as in antisense treatment. Samples were processed for cell cycle analysis at days 2 and 7 following treatment with 20 mM oligonucleotides, by ®xation with 70% ethanol and redilution in a mixture of propidium iodide and RNase. Cell cycle pro®le was determined by ¯ow cytometry (Epics XL-MCL, Coulter) analyzing at least 10 000 cells. 2.9. Statistical analysis Differences between ASO and SO treatments were analyzed using a paired t-test (StatView 4.02 for MacIntosh). 3. Results 3.1. Growth suppression of CML cell lines after individual treatment with ASOs In an attempt to determine if the bcr-abl ASO was speci®c for the b2a2 translocation for which it had been designed [1], 20 mM ASO or SO were added to triplicate cultures of BV173 (Fig. 1a), K562 (Fig. 1b) or HL60 (Fig. 1c) cells in culture on day 1. Half of the doses were added on day 2 and a fourth on day 3. In the BV173 cell line, with the b2a2 translocation, the ASO reduced viable cells by nearly 40% on day 9, as compared with the control oligonucleotide or with no treatment (Fig. 1a). In the K562 cell line (with a b3a2 translocation) and in the HL60 cell line (neither b2a2 nor b3a2 translocation) there was no difference
Fig. 1. Effect of bcr-abl ASO or SO in the viable cell number of different cell lines. BV173 cells (starting concentration 5 £ 10 5/ml) (a), K562 cells (2 £ 10 5/ml) (b) or HL60 cells (2 £ 10 5/ml) (c) were treated with 20 mM ASO or SO or with control media at day 1, 10 mM at 24 h and 5 mM at 48 h. Viable cells were counted at days 4, 7 and 9. Results are the mean of three independent experiments (three replicates each). * Represents P # 0:05 between AS and control treatments and 1 represents P # 0:05 between S and control treatments.
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when compared with control cells. In the BV173 cell line such cytotoxicity was not found at day 9. A study was performed on BV173 by comparing the response of cells treated as above with cells treated with half dose, that is 10 mM on day 1, 5 mM on day 2 and 2.5 mM on day 3. Table 1 represents the data obtained on day 7 of treatment. At 10 mM there was no cell death caused either by the sense or the antisense oligonucleotides. The effect of the TfR ASO was also tested in the BV173 cell line, under the same treatment conditions as the bcr-abl ASO. The ASO reduced the number of viable cells by nearly 30% on day 9, as compared with no treatment (Fig. 2). However, the SO also reduced viable cell number, at this concentration. In order to reduce this cell death, a study was performed at reduced oligo concentration as for the bcr-abl oligonucleotides. The TfR SO was less cytotoxic at 10 mM than at 20 mM (Table 1). However, as in the case of bcr-abl, the TfR ASO is less effective at 10 mM than at 20 mM, if compared with the SO treatment. 3.2. Growth suppression of CML cell lines following concomitant treatment with both ASOs A combined treatment with both oligonucleotides was also carried out, using a 20 mM concentration of Table 1 Number of viable cells obtained by individual or concomitant ASO treatment of BV173 for 7 days a Treatment
Number of % of SO ^ SE (n) viable cells (£10 5/ml) ^ SE (n)
Control (medium only) bcr-abl ASO bcr-abl SO TfR ASO TfR SO bcr-abl 1 TfR ASO bcr-abl 1 TfR SO
41.6 ^ 3.1 37.7 ^ 4.3 40.0 ^ 5.5 35.7 ^ 4.5 39.5 ^ 6.8 27.7 ^ 5.5 37.0 ^ 3.6
(3) (3) (3) (3) (3) (3) (3)
± 95 ^ 7 (3) ± 92 ^ 5 (3) ± 75 ^ 12 (3) ±
a Cultures of BV173 cells (5 £ 10 5/ml) were treated with bcr-abl or TfR or with a combination of both bcr-abl and TfR oligonucleotides. Treatments were with 10 mM of each oligonucleotide individually or with a combination of both oligonucleotides (5 mM of each). These concentrations of oligonucleotides were added at day 1 and were followed by treatment with half the dose at 24 h and a quarter of the dose at 48 h. Results are the mean of three independent experiments (three replicates each).
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Fig. 2. Effect of TfR ASO or SO in the viable cell number of BV173 cells. Cells (5 £ 10 5/ml) were treated with 20 mM ASO or SO or with control media at day 1, 10 mM at 24 h and 5 mM at 48 h. Viable cells were counted at days 4, 7 and 9. Results are the mean of three independent experiments (three replicates each). * Represents P # 0:05 between AS and control treatments.
each oligonucleotide (total of 40 mM oligonucleotides concentration). Data proved that this treatment was very toxic to the cells as cell death was very high (results not shown). Using half the concentration of each oligonucleotide (10 mM each therefore a total of 20 mM oligonucleotide concentration) there was still toxicity caused by the sense oligonucleotide treatments. At this concentration, the ASO treatment reduced cell number by 22% (P 0:03) when compared with SO treatment (results not shown). Reducing the dose of the oligonucleotide treatment even further to 5 mM each (total of 10 mM oligonucleotide concentration) there was no toxic effect caused by the sense treatment and the concomitant antisense treatment reduced cell number by 25% when compared with the concomitant SO treatment, as compared with 5% reduction obtained with the bcrabl ASO and 8% reduction obtained with the TfR ASO at 10 mM (Table 1) in relation to the respective SO controls. Thus, the 10 mM synergistic treatment was more ef®cient in reducing cell growth in the BV173 cell line than the individual ASO treatments. 3.3. Speci®city of action To determine whether the bcr-abl ASO was speci®c towards its own mRNA, BV173 cells were treated with 20 mM AS or S oligonucleotides for 24 or 48 h, at which time points total mRNA was extracted and transferred into a membrane which was further probed
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with a bcr-abl cDNA speci®c for the b2a2 translocation. To compensate for differences in loading the membrane was reprobed for the GAPDH mRNA. Results indicate that there was only a slight reduction (ASO compared to SO treatment) of the bcr-abl mRNA at 24 h (8% reduction) and 48 h (11% reduction) following oligonucleotide treatment (Fig. 3a). Further to this study, the BCR-ABL protein levels were determined by Western blotting (Fig. 3b) 48, 72 and 96 h following treatment with either ASO or
SO. These results show a reduction of BCR-ABL (34% at 48 h and 15% at 72 and 96 h) when comparing the ASO with the SO treatment. The speci®city of the TfR ASO was studied by ¯ow cytometric analysis of CD711 cells. BV173 cells were treated with 20 mM ASO or SO for 48 h, at which time the cells were processed for ¯ow cytometric analysis, labeling them with a TfR antibody. Results (Fig. 4) indicated that 20 mM TfR ASO were capable of reducing TfR expression 48 h following treatment, as compared with the SO, by 24%. However, the SO also seemed to reduced the expression of TfR, when compared with the control cells, by nearly 20%. This correlates well with the cytotoxic effect detected for the TfR SO at a later time (Fig. 2). 3.4. Analysis of the cell cycle Studies of the cell cycle distribution by ¯ow cytometry revealed that either the individual treatments with ASOs or the concomitant treatment with both oligonucleotides (20 mM) caused an accumulation of cells in S phase by day 7, preventing cells from progressing in the cell cycle (Fig. 5). Furthermore, it was possible to observe a sub-G1 peak, which was bigger in the concomitant treatment with the two ASOs than in the individual treatments, suggesting an increase in apoptosis by day 7.
Fig. 3. Speci®city of the bcr-abl antisense effect. Effect at the mRNA level (a): Northern Blot of BV173 cells (10 £ 10 5/ml) treated with bcr-abl ASO or SO or with medium (no oligos). RNA was extracted at 24 h after treatment with 20 mM oligonucleotide or at 48 h after treatment with 20 mM oligonucleotide at day 1 and 10 mM at 24 h. The membrane was probed with a cDNA obtained by RTPCR and reprobed with GAPDH cDNA. Effect at the protein level (b): Western Blot of BV173 cells (10 £ 10 5/ml) treated with bcr-abl ASO or SO or with medium alone. Proteins were extracted at 48, 72 and 96 h after treatment with 20 mM oligonucleotide, 10 mM at 24 h and 5 mM at 48 h. The membrane was probed with an anti-c-abl primary antibody and reprobed with an actin antibody. Results shown in the text are comparison of bcr-abl/actin ratios in the different conditions.
Fig. 4. Speci®city of the TfR antisense effect. Flow cytometric analysis of TfR protein expression in BV173 cells 48 h after incubation with 20 mM oligonucleotide and 10 mM at 24 h.
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Fig. 5. Representative example of the effect of antisense treatment in the cell cycle pro®le. BV173 cells were treated with bcr-abl or TfR oligonucleotides or with both (with half the dose of each oligonucleotide). The total oligonucleotide added was 20 and 10 mM at 24 h and 5 mM at 48 h. Cell cycle pro®le was analyzed at days 2 and 7 after treatment.
4. Discussion Treatment with antisense oligonucleotides, on their own or combined with other cytotoxic agents, is a promising strategy in cancer therapy, mainly in stages of minimal disease. Diseases which have unique genes resulting from non-random chromosomal translocations are likely targets and this approach has been tested by basic and clinical researchers for the past few years. Amongst these fusion genes the most commonly tested has been the bcr-abl gene in CML [1,2,18]. However treatment with bcr-abl ASO has been proved so far insuf®cient to kill all malignant cells. In the present work, we have attempted to improve the speci®c killing capacity of ASO towards bcr-abl by concomitant treatment with ASO towards the TfR gene. Although TfR is ubiquitously expressed we
believe that a selective effect could be obtained due to a higher need of the malignant cells for iron. We designed a new ASO for TfR, which reduced BV173 viable cell number by nearly 30% at 20mM concentration, also decreasing TfR protein levels. The bcr-abl ASO used was the one designed by Szczylik et al. [1], which reduced BV173 viable cell number by 40% at 20 mM concentration in our hands. Similar results had been obtained by others, using the same oligonucleotide and the same concentration [18]. We found evidence for a small reduction of bcr-abl mRNA and protein levels; furthermore, this bcr-abl ASO was speci®c for the b2a2 translocation of the bcr-abl gene, as demonstrated by ASO treatment of control cell lines (K562 and HL60). This indicates that this ASO would be able to target the bcr-abl mRNA of malignant cells, without causing normal cell death.
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The TfR SO presented some toxicity to the cells at 20 mM but not at 10 mM concentration. In agreement to this, the less cytotoxic concomitant oligonucleotide treatment was the one carried out at 10 mM (total oligonucleotide concentration). Indeed, at this concentration the concomitant SO treatment did not cause cytotoxicity, whereas the concomitant ASO treatment reduced viable cell number by 25% when compared with the SO, 7 days after the beginning of the treatment. This reduction was greater than the one obtained by the individual use of the ASO (220 and 217% than bcr-abl ASO and TfR ASO alone) at 10mM concentration, thus proving the existence of a synergistic effect. When the non-speci®c toxicity of the oligonucleotides is reduced the antisense effect becomes more evident. The ef®cacy and speci®city of the oligonucleotides were also proved at a molecular level, by reducing the levels of the corresponding mRNAs or proteins. However, reduction in bcr-abl mRNA and protein levels was not as pronounced as one would expect for two possible reasons: (i) the ASO is not very ef®cient; (ii) the time points studied were not the ones at which levels of mRNA and protein are the lowest. Furthermore, both the individual ASO treatments and the concomitant treatment prevented progression in the cell cycle and increased the sub-G1 peak suggestive of apoptosis. This was more pronounced in the concomitant treatment with the two ASOs than in the individual treatments. Thus, we conclude that this approach might be useful in controlling proliferation of CML cells since even though we did not obtain a very strong reduction in viable cell number, all the results indicate that the bcr-abl ASO is speci®c to bcr-abl mRNA and that there is a synergism of action of both ASOs when used concomitantly. Better reduction of viable cell number would be expected if less toxic and more speci®c and potent oligonucleotides could be designed. In recent years several chemical modi®cations of oligonucleotides that improve uptake and potency without increasing toxicity have been designed (reviewed in [12]). Alternatively, liposomes [19,20] or Streptolysin O [21,22] may be used to improve cellular uptake of oligonucleotides, therefore increasing ef®ciency. We are currently investigating these possibilities of improving antisense oligonucleotide ef®cacy.
Acknowledgements We thank Professor C. Szczylik for proof-reading this manuscript and Dr Filipe Sansonetty's team (IPATIMUP) for analysis on the Flow Cytometer. This work was supported by FCT (Portugal), project PRAXIS/2/2.1/SAU/1283/95. Sandra Beleza has a BIC grant and M. Helena Vasconcelos has a postdoctoral grant from FCT. References [1] C. Szczylik, T. Skorski, N.C. Nicolaides, L. Manzella, L. Malaguarnera, D. Venturelli, A.M. Gewirtz, B. Calabretta, Selective inhibition of leukemia cell proliferation by BCRABL antisense oligodeoxynucleotides, Science 253 (1991) 562±565. [2] T. Skorski, M. Nieborowska-Skorska, N.C. Nicolaides, C. Szczylik, P. Iversen, R.V. Iozzo, G. Zon, B. Calabretta, Suppression of Philadelphia leukemia cell growth in mice by BCR-ABL antisense oligodeoxynucleotide, Proc. Natl. Acad. Sci. USA 91 (1994) 4504±4508. [3] T.F.C.M. Smetsers, T. Skorski, L.T.F. van de Locht, H.M.C. Wessels, A.H.M. Pennings, T. de Witte, B. Calabretta, E.J.B. Mensink, Antisense BCR-ABL oligonucleotides induce apoptosis in the Philadelphia chromosome-positive cell line BV173, Leukemia 8 (1) (1994) 129±140. [4] A. McGahon, R. Bissonnette, M. Schmitt, K.M. Cotter, D.R. Green, T.G. Cotter, BCR-ABL maintains resistance of chronic myelogenous leukemia cells to apoptotic cell death, Blood 83 (5) (1994) 1179±1187. [5] G. Anfossi, A.M. Gewirtz, B. Calabretta, An oligomer complementary to c-myb-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines, Proc. Natl. Acad. Sci. USA 86 (1989) 3379±3383. [6] B. Calabretta, R.B. Sims, M. Valtieri, D. Caracciolo, C. Szczylik, D. Venturelli, M. Ratajczak, M. Beran, A.M. Gewirtz, Normal and leukemic hematopoietic cells manifest differential sensitivity to inhibitory effects of c-myb antisense oligodeoxynucleotides: an in vitro study relevant to bone marrow purging, Proc. Natl. Acad. Sci. USA 88 (1991) 2351±2355. [7] R. Heikkila, G. Schwab, E. Wickstrom, S.L. Loke, D.H. Pluznik, R. Watt, L.M. Neckers, c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G0 to G1, Nature 328 (1987) 445±449. [8] G. Degols, J.-P. Leonetti, N. Mechti, B. Lebleu, Antiproliferative effects of antisense oligonucleotides directed to the RNA of c-myc oncogene, Nucleic Acids Res. 19 (4) (1991) 945± 948. [9] T. Skorski, D. Perrotti, M. Nieborowska-Skorska, S. Gryaznov, B. Calabretta, Antileukemia effect of c-myc N3 0 -P5 0 phosphoramidate antisense oligonucleotides in vivo, Proc. Natl. Acad. Sci. USA 94 (1997) 3966±3971.
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