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Clinical Cancer Research Vol. 6, 2547-2555, June 2000
© 2000 American Association for Cancer Research


Experimental Therapeutics, Preclinical Pharmacology

A Novel Bispecific Antisense Oligonucleotide Inhibiting Both bcl-2 and bcl-xL Expression Efficiently Induces Apoptosis in Tumor Cells1

Uwe Zangemeister-Wittke2, Siân H. Leech, Robert A. Olie, A. Paula Simões-Wüst, Oliver Gautschi, Gerd H. Luedke, François Natt, Robert Häner, Pierre Martin, Jonathan Hall, Carlo M. Nalin and Rolf A. Stahel

Division of Oncology, Department of Internal Medicine, University Hospital Zürich, CH-8044 Zürich, Switzerland [U. Z-W., S. H. L., R. A. O., A. P. S-W., O. G., G. H. L., R. A. S.]; Functional Genomics Area, Novartis Pharma AG, CH-4002 Basel, Switzerland [F. N., R. H., P. M., J. H.]; and Novartis Institute for Biomedical Research, Novartis Pharma Inc., Summit, New Jersey 07901-1398 [C. M. N.]


    ABSTRACT
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bcl-2 and Bcl-xL are inhibitors of apoptosis frequently overexpressed in solid tumors. The bcl-2 and bcl-xL mRNAs share a region of homology comprising nucleotides 605–624 and 687–706, respectively, which differs by only three nucleotides. This sequence does not occur in the proapoptotic splice variant bcl-xS. To test the possibility that oligonucleotides targeting this region have the potential to down-regulate bcl-2 and bcl-xL expression simultaneously, three 2'-O-methoxy-ethoxy-modified phosphorothioate oligonucleotides were designed. These oligonucleotides differed in the number of mismatches to bcl-2 and bcl-xL and in the number of nucleotides to which the modifications were made. The effects of these oligonucleotides on bcl-2 and bcl-xL expression, as well as their abilities to induce apoptosis, were assessed in small cell and non-small cell lung cancer cell lines expressing different basal levels of bcl-2 and bcl-xL. Although all oligonucleotides down-regulated bcl-2 and bcl-xL expression, oligonucleotide 4625, which has no mismatching nucleotides to bcl-2 but three to bcl-xL, two of which were modified by 2'-O-methoxy-ethoxy residues, showed the strongest bispecific activity on the transcript and protein level. In all cell lines this bispecific activity induced apoptotic cell death, as demonstrated by increased uptake of propidium iodide, a 10–100-fold increase in caspase-3-like protease activity, and nuclear condensation and fragmentation. This is the first report of a bcl-2/bcl-xL bispecific antisense oligonucleotide that deserves attention as a therapeutic compound in lung cancer and other malignancies in which bcl-2 and/or bcl-xL are overexpressed.


    INTRODUCTION
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Bcl-2 family of proteins includes antiapoptotic and proapoptotic members that are differentially regulated, but share sequence-homology domains. Pro- and antiapoptotic family members are able to heterodimerize, thereby modulating one another’s function. It has been suggested that their relative concentrations may function as a rheostat for the apoptotic program (1) . Up-regulated expression of the antiapoptotic proteins Bcl-2 and/or Bcl-xL has been found in various tumors. Recent findings suggest that both proteins confer drug resistance by abrogating mitochondrial cytochrome c release and activation of caspase-3 (2) . Although Bcl-2 and Bcl-xL are considered to be functionally indistinguishable, there is evidence to support distinct biological roles of these proteins for protection from apoptosis induced by different cytotoxic stimuli (3 , 4) . This issue is further complicated by the cellular heterogeneity of tumor tissues and the finding that tumor cells may switch expression from one death antagonist to the other (5) .

Antisense oligonucleotides are useful as tools for biological research, target validation, and as drugs to inhibit disease-related gene expression (6) . Antisense oligonucleotides inhibiting bcl-2 or bcl-xL expression could prove to be potent inducers of apoptosis and to be of therapeutic benefit for various hyperproliferative diseases, including cancer (7, 8, 9, 10) . On the basis of the uncertainty about which of the two genes might be of greater relevance for cell survival in a given tumor, the use of antisense compounds that inhibit both bcl-2 and bcl-xL expression simultaneously could be of therapeutic benefit. The most challenging issue in antisense design is the lack of mRNA sites that can be targeted efficiently, and several in vitro techniques have been used to predict antisense efficacy (11) . In the present study, we used an algorithm combining sequence alignment (12) with prediction of mRNA sequences, presented in single-strand conformation by use of the RNAdraw program (13) , to design 2'-O-methoxy-ethoxy-modified phosphorothioate antisense oligonucleotides (14) targeting a region of high homology shared between the bcl-2 and the bcl-xL mRNAs. Such second generation antisense compounds are superior to their first generation "deoxy" counterparts with regard to stability and target hybridization affinity and also show a lower degree of unspecific toxicity (6 , 15 , 16) . As the most promising target sequence, a region encompassing the splice junction site of the bcl-x gene was identified. This sequence does not occur in mRNAs of the proapoptotic bcl-2 family members bax, bak, or bcl-xS, and in the bcl-x pre-mRNA it is interrupted by an intron. Examination of three potentially bcl-2 and bcl-xL bispecific antisense oligonucleotides by use of small cell and non-small cell lung cancer cell lines revealed one compound, oligonucleotide 4625, that most efficiently inhibited the expression of both antiapoptotic genes and induced tumor cell apoptosis.


    MATERIALS AND METHODS
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor Cells.
The small cell lung cancer cell line SW2 was obtained from Dr. S. D. Bernal (Dana-Farber Cancer Institute, Boston, MA). The non-small cell lung cancer cell lines NCI-H125 and A549 were obtained from the American Type Culture Collection (Manassas, VA). Tumor cells were cultured in RPMI 1640 (Hyclone Europe, Ltd., Cramlington, United Kingdom) supplemented with 2 mM L-glutamine, 10% FCS (Hyclone Europe, Ltd.), 50 IU/ml penicillin, and 50 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2.

Antisense Oligonucleotides.
Antisense oligonucleotides were derived from sequence alignment by use of the Macaw software (12) to identify regions of high homology between bcl-2 and bcl-xL and the RNAdraw program (13) to predict sequences possibly presenting in a single-stranded conformation. On the basis of our experience from a bcl-2 antisense gene walk study comprising a series of different antisense sequences (7) , the use of the RNAdraw program is useful to predict potentially accessible hybridization sites on the target mRNA and to limit the overall number of target sites to be tested. All oligonucleotides were 20-mers with a phosphorothioate backbone and two to five nucleotides at the 5' and 3' ends modified by MOE3 residues at the 2'-{alpha}-position of the deoxy ribose (represented by underlined letters in the sequences shown below). Oligonucleotides were synthesized, as described (14) , on an Oligopilot II (Amersham Pharmacia Biotech, Uppsala, Sweden) at 150–180 µmol scale on Primer polystyrene (Amersham Pharmacia Biotech) derivatized via succinyl arm by the first corresponding 2'-O-methoxy-ethoxy 3' nucleoside. Crude oligonucleotides were purified by reverse-phase high-pressure liquid chromatography. Purity was assessed by Capillary Gel Electrophoresis, phosphodi-ester content was assessed by 31P-NMR, and the oligonucleotides were characterized by Maldi-Tof mass spectrometry. All oligonucleotides displayed a length purity higher than 95% with a phosphodi-ester content lower than 0.3%. The antisense sequences were as follows:

4625, 5'-AsAsGsGsCsAsTsCsCsCsAsGsCsCsTsCsCsGsTsT-3';

4627, 5'-AsAsAsGsCsAsTsCsCsCsAsGsCsCsTsCsCsGsTsT-3';

4259, 5'-AsAsAsGsTsAsTsCsCsCsAsGsCsCsGsCsCsGsTsT-3'.

In addition to these three antisense oligonucleotides, oligonucleotide 4626 with the sequence 5'-CsAsCsGsTsCsAsCsGsCsGsCsGsCsAsCsTsAsTsT-3' was used as a scrambled control of oligonucleotide 4625.

Delivery of Antisense Oligonucleotides to Tumor Cells.
Oligonucleotides were delivered to cells in the form of complexes with the transfection reagent lipofectin, a 1:1 (w/w) formulation of lipids containing N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride and dioleoyl phosphotidylethanolamine (Life Technologies, Inc., Glasgow, United Kingdom). Briefly, lipofectin (100 µg/ml) was allowed to complex with oligonucleotides (6.5 µM) in serum- and antibiotic-free medium, before dilution and addition to cells. Following various times of incubation in cultures with SW2 cells growing in suspension, fresh medium (containing FCS, glutamine, and antibiotics) was added to the transfection medium to dilute out the transfection reagent, whereas in cultures with adherently growing NCI-H125 and A549 cells medium was replaced completely. Cells were further incubated at 37°C in a humidified atmosphere containing 5% CO2.

Real-Time PCR.
Total RNA was isolated from cells by use of the RNeasy Mini Kit (Qiagen AG, Basel, Switzerland). For cDNA synthesis, Taqman Reverse Transcription Reagents were used as described in the user’s manual of the ABI Prism 7700 Sequence Detection system with which real-time PCR amplification was performed according to the Taqman Universal PCR Master Mix protocol (Perkin-Elmer Applied Biosystems, Foster City, CA). Relative quantification of gene expression was performed as described in the manual using rRNA as an internal standard and the comparative CT (threshold cycle) method. Bcl-2 and bcl-xL cDNAs were amplified using primers and Taqman probes that had been selected with the Primer Express Applications-Based Primer Design Software (Perkin-Elmer Applied Biosystems). For bcl-xL, primers with the sequences 5'-TCCTTGTCTACGCTTTCCACG-3' and 5'-GGTCGCATTGTGGCCTTT-3' were used together with a 5'-ACAGTGCCCCGCCGAAGGAGA-3' Taqman probe. For bcl-2, primers with the sequences 5'-CATGTGTGTGGAGAGCGTCAA-3' and 5'-GCCGGTTCAGGTACTCAGTCA-3' were used together with a 5'-CCTGGTGGACAACATCGCCCTGT-3' Taqman probe. The bcl-xL and bcl-2 probes were labeled at the 5' end with the reporter dye molecule 6-carboxy-fluorescein and at the 3' end with the quencher dye molecule 6-carboxy-tetramethyl-rhodamine. A Basic Local Alignment Search Tool search of the National Center for Biotechnology Information database revealed no homology of the primer and probe sequences to other known human genes. Relative data are presented in comparison with an untreated control sample chosen as calibrator. The range of a given sample relative to the calibrator was always <10%. All data reported are based on determinations done at least in duplicates.

Western Blot Analysis.
Lysates from cells treated with oligonucleotides were subjected to Western blot analysis as described (7) . Lysates from cells treated with oligonucleotides in the presence of the caspase inhibitor zVAD.fmk (Bachem, Dübendorf, Switzerland) were used as controls to exclude the possibility that changes in Bcl-2 and Bcl-xL levels were due to caspase-mediated degradation (17 , 18) . Briefly, 20 µg of soluble protein per sample were separated on 12% polyacrylamide SDS gels. Transfer to polyvinylidene fluoride membranes was performed in semi-dry blotting chambers for 1 h. The blots were blocked in Tris-buffered saline containing 5% bovine serum albumin and 5% nonfat milk and then incubated overnight at 4°C with mouse antihuman Bcl-2 monoclonal antibody (DAKO Diagnostics AG, Glostrup, Denmark) or rabbit antihuman Bcl-xL monoclonal antibody (Transduction Laboratories, Lexington, KY). Actin staining with a mouse antiactin monoclonal antibody (ICN Biomedicals, Inc., Aurora, OH) was used as a loading control. To detect the primary antibodies, blots were incubated with rabbit antimouse or goat antirabbit immunoglobulin peroxidase conjugates (Sigma Chemical Co., St. Louis, MO) for 1 h at room temperature. Visualization of the immunocomplexes was performed by enhanced chemiluminescence using the ECL kit (Amersham Pharmacia Biotech, Dübendorf, Switzerland), followed by exposure to X-ray films. Relative protein levels were quantified using Scion software (Scion Corporation, Frederick, MD) on scanned films.

Determination of Tumor Cell Death.
Tumor cell death was determined based on PI uptake by use of a FACScalibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA). Cells were incubated with 0.5 µg/ml PI for 5 min at room temperature and washed with PBS, and the fraction of cells with increased fluorescence intensity was measured. Cell debris was excluded from analysis by appropriate light scatter gating.

Measurement of Caspase-3-like Protease Activity.
Caspase-3-like protease activity in cell lysates was analyzed in a colorimetric assay. Cells were lysed in buffer by two freeze/thaw cycles essentially as described (19) , and lysates were centrifuged at 14,000 rpm (17,500 x g) at 4°C for 15 min. Soluble cytosolic protein (40 µg) was mixed with 80 µM of the caspase-3-specific substrate DEVD-pNa (Bachem, Dübendorf, Switzerland) in a final volume of 100 µl and incubated at 37°C. Subsequently, substrate cleavage was monitored at 405 nm using a SPECTRAmax 340 microplate reader and analyzed using SOFTmax PRO software (Molecular Devices, Sunnyvale, CA). To confirm that substrate cleavage was due to caspase activity, extracts were incubated in the presence of 10 µM of the caspase-3-specific inhibitor DEVD-CHO (Bachem) for 30 min at 37°C, before the addition of substrate. The absorbance signal (in arbitrary units) of the DEVD-CHO-inhibited sample was subtracted from the absorbance signal of the uninhibited sample.

Hoechst Staining of Cells.
Cells were washed with PBS and fixed for 15 min in 5 µg/ml Hoechst dye-containing 4% paraformaldehyde/0.05% saponin. Subsequently, cells were washed three times with PBS, centrifuged onto glass slides by cytospin centrifugation, and mounted with Mowiol. Photographs were taken by use of a Leica confocal laserscan fluorescence microscope (Leitz, Wetzlar, Germany).


    RESULTS
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Design of Bispecific Antisense Oligonucleotides.
Alignment of the sequences of bcl-2 and bcl-xL reveals a region of high homology comprising nucleotides 605–624 and 687–706 of the bcl-2 and bcl-xL mRNAs, respectively. This region encompasses the splice junction site of bcl-x and, thus, does not occur in the pre-mRNA and is largely deleted in the proapoptotic splice variant bcl-xS. In this region, an antisense oligonucleotide would be expected to hybridize to both the bcl-2 and bcl-xL mRNAs, if it tolerates a maximum of three mismatches. Moreover, use of the RNAdraw program revealed that in both mRNA species this region possibly exists mainly in single-stranded form. To test the hypothesis that this sequence might be an accessible binding site for oligonucleotides, we have designed three 20-mer oligonucleotides. The sequence alignment of the antisense oligonucleotides and the bcl-2 and bcl-xL mRNAs is shown in Fig. 1Citation . A Basic Local Alignment Search Tool for Nucleotides search of a database containing all sequences of GenBank, European Molecular Biology Laboratory, DNA Data Base of Japan, and Protein Data Base revealed no homology of the oligonucleotides to other human genes. Furthermore, the hybridization affinity and nuclease resistance of the oligonucleotides was enhanced by MOE modifications of selected riboses at the 5' and 3' ends. Oligonucleotide 4625 has 100% sequence identity to bcl-2 and three mismatches to bcl-xL; oligonucleotide 4627 has one mismatch to bcl-2 and two mismatches to bcl-xL. The numbers and types of mismatching bases as well as the hybridization sites in the bcl-2 and bcl-xL mRNAs are shown in Table 1Citation . In oligonucleotide 4625 the MOE modifications included the bcl-xL mismatching nucleotides at positions 3 and 5. In oligonucleotide 4627 the MOE modifications included the bcl-2 mismatching nucleotide at position 3 and the bcl-xL mismatching nucleotide at position 5. Oligonucleotide 4259 was designed with 100% sequence identity to bcl-xL and three mismatching nucleotides to bcl-2. With the aim to maintain its preferential specificity for bcl-xL, these three mismatches were not subjected to MOE modifications.



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Fig. 1. Alignment of oligonucleotides 4625, 4627, and 4259 and the bcl-2 and bcl-xL mRNAs by use of the Macaw software (12) . For clarity, only parts of the mRNA sequences are shown. The exact hybridization sites of the oligonucleotides in the target mRNAs are given in Table 1Citation . Sequence identity to bcl-2 is depicted by black boxes. The white insets represent mismatches to bcl-2.

 

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Table 1 Hybridization sites of the antisense oligonucleotides in the bcl-2 and bcl-xL mRNAs

 
Down-Regulation of bcl-2 and bcl-xL mRNAs in Lung Cancer Cells.
To measure the ability of the antisense oligonucleotides 4625, 4627, and 4259 to bind and induce the degradation of the bcl-2 and bcl-xL mRNAs, real-time PCR analyses of the transcripts were performed. For this purpose, the SW2 cell line was chosen because it expresses similar and easily detectable levels of bcl-2 and bcl-xL. In addition to the three antisense compounds, which themselves serve as mismatch controls for each other, oligonucleotide 4626 was used as a scrambled sequence control of oligonucleotide 4625. Fig. 2Citation shows the bcl-2 and bcl-xL mRNA levels following a 6-h treatment with the different oligonucleotides at doses of 300-1200 nM. Increasing the dose from 300 nM to 600 nM increased the activity of the oligonucleotides on both target mRNAs, with the exception of oligonucleotide 4259 on bcl-xL, which showed equal activity at 300 and 600 nM. Oligonucleotide 4625 revealed the strongest bispecific effect, followed by oligonucleotide 4627, whereas oligonucleotide 4259 preferentially down-regulated the bcl-xL mRNA. Further increasing the dose of the oligonucleotides to 1200 nM did not result in increased antisense activity and started to become unspecifically toxic to the cells in proliferation assays (data not shown). Therefore, in all additional experiments an oligonucleotide dose of 600 nM was used.



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Fig. 2. Bcl-2 and bcl-xL mRNA levels in SW2 cells after treatment with different doses of antisense oligonucleotides. Cells were treated with 300 nM or 600 nM oligonucleotides, and mRNA levels were quantified 6 h after the start of treatment by real-time PCR analysis using the ABI Prism 7700 Sequence Detection System, as described under "Materials and Methods." Data were normalized by use of rRNA as an internal standard, following confirmation that bcl-2, bcl-xL, and rRNA were amplified with the same efficiency. Data are presented relative to untreated cells; bars, SD.

 
To investigate whether the antisense effects of the oligonucleotides were time dependent, bcl-2 and bcl-xL mRNA levels were quantified at various time points following the start of treatment with 600 nM oligonucleotides. As shown in Fig. 3, A and BCitation , during a 6-h transfection, oligonucleotides 4625 and 4627 reduced bcl-2 and bcl-xL mRNA levels to approximately 23% and 40%, and 45% and 57%, respectively, of initial values. Oligonucleotide 4259 preferentially reduced bcl-xL mRNA levels to 14% and showed little activity against bcl-2. The different specificities and effects of the oligonucleotides became even more obvious when measured 20 h after the start of transfection (Fig. 3C)Citation . As shown above for a 6-h treatment, also at this later time point an increase in the dose of oligonucleotides from 600 nM to 1200 nM did not result in increased antisense activity (data not shown). These findings identified oligonucleotide 4625 as the most potent bcl-2/bcl-xL bispecific antisense compound among the three oligonucleotides tested.



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Fig. 3. Bcl-2 and bcl-xL mRNA levels in SW2 cells at different time points after the start of treatment with 600 nM antisense oligonucleotides. For mRNA quantification, real-time PCR analysis was performed as described in the legend to Fig. 2Citation . A and B, relative amounts of bcl-2 and bcl-xL mRNA during the first 6 h after the start of treatment. Data are presented relative to cells treated under the same conditions and harvested immediately after the addition of the oligonucleotides (0 h); bars, SD. C, relative amounts of bcl-2 and bcl-xL mRNA 20 h after the start of treatment. Data are presented relative to untreated cells; bars, SD.

 
Down-Regulation of Bcl-2 and Bcl-xL Protein in Lung Cancer Cells.
Having demonstrated the differential specificities of the antisense oligonucleotides 4625, 4627, and 4259 on the mRNA level, their abilities to down-regulate Bcl-2 and Bcl-xL protein levels in SW2 cells were examined by Western blot analysis. Oligonucleotide 4626 was used as a scrambled sequence control of the most potent bispecific antisense compound 4625. Fig. 4Citation shows that all three antisense oligonucleotides reduced Bcl-2 and Bcl-xL levels in target cells, although to different extents, as expected from their effects on the respective mRNA. Forty h after the start of transfection, oligonucleotide 4625 showed the strongest effect and reduced Bcl-2 levels to 22% of the untreated control value. The effect of oligonucleotide 4627 was less pronounced (reduction to 39%), and oligonucleotide 4259 had the lowest down-regulating activity (reduction to 52%). On Bcl-xL expression the strongest effects were mediated by oligonucleotides 4625 and 4259, which both reduced protein levels to 18% of the untreated control value. Oligonucleotide 4627 reduced Bcl-xL levels to 24%. These effects on Bcl-2 and Bcl-xL were not due to antisense-unrelated degradation of the proteins mediated by caspases because a comparable pattern of down-regulation was observed after treatment of cells in the presence of the caspase inhibitor zVAD.fmk. As expected, the antisense oligonucleotides did not reduce the marginal level of Bcl-xS detectable in SW2 cells (data not shown). Thus, the antisense effects determined on the protein levels correlated well with those observed on the mRNA levels shown in Figs. 2Citation and 3Citation and again suggest oligonucleotide 4625 as the most potent bcl-2/bcl-xL bispecific antisense compound.



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Fig. 4. Western blot analysis of Bcl-2 and Bcl-xL expression in SW2 after treatment with antisense oligonucleotides. Cells were treated without (Untreated, Lipofectin) or with 600 nM oligonucleotides, and protein expression was assessed 40 h after the start of treatment. Blots were reprobed for ß-actin to confirm equal protein loading. Values represent the percentage of protein expression quantified using Scion software on scanned films and corrected for ß-actin loading.

 
Induction of Death in Lung Cancer Cells.
Analysis of the different antisense oligonucleotides on the transcript and protein level identified oligonucleotide 4625 as the most potent bcl-2/bcl-xL bispecific antisense compound. To investigate whether the simultaneous down-regulation of Bcl-2 and Bcl-xL expression by use of oligonucleotide 4625 induces death in small cell and non-small cell lung cancer cells, their uptake of PI was examined by FACS analysis 40 and 64 h after the start of treatment with 600 nM oligonucleotides. Oligonucleotide 4259, which preferentially down-regulates Bcl-xL expression, was tested for comparison. As shown in Fig. 5Citation , the bispecific oligonucleotide 4625 showed a strong cytotoxic effect on all three cell lines and increased the number of dead cells from 25% to 33%, as compared with about 5% in untreated control cultures. FACS analysis of antisense-treated cells revealed a large amount of cell debris, which was excluded from PI uptake analysis. Thus, due to their rapid disintegration, a substantial fraction of dead cells was not detectable at the time of analysis, indicating that the actual number of dead cells in the cultures was even higher than measured. Interestingly, treatment with oligonucleotide 4259 was not cytotoxic to SW2 cells, whereas it reduced the viability of NCI-H125 and A549 cells to comparable levels as oligonucleotide 4625.



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Fig. 5. Death of lung cancer cells after treatment with antisense oligonucleotides. SW2, NCI-H125, or A549 cells were treated without (Untreated, Lipofectin) or with 600 nM oligonucleotides, and cell death was determined based on PI uptake measured by FACS analysis 40 and 64 h after the start of treatment. Representative results of one of three independent experiments are shown.

 
Induction of Apoptosis in Lung Cancer Cells.
To demonstrate that the induction of death in lung cancer cells following treatment with oligonucleotide 4625 (shown in Fig. 5Citation ) was due to the induction of apoptosis, caspase-3-like protease activity, and nuclear condensation and fragmentation were examined 40 and 64 h after the start of treatment with 600 nM of this oligonucleotide. Oligonucleotide 4259 was tested for comparison. Fig. 6Citation shows that in all three cell lines treatment with oligonucleotide 4625 resulted in a strong increase in caspase activity, which was about 10-fold in SW2 cells, 15-fold in NCI-H125 cells, and 100-fold in A549 cells as compared with the respective untreated controls. Oligonucleotide 4259, which preferentially down-regulates Bcl-xL expression, did not increase caspase activity in SW2 cells but increased caspase activity in NCI-H125 and A549 cells about 6-fold and 7-fold, respectively. This again indicates that compared with SW2 cells the non-small cell lung cancer cell lines were more prone to apoptosis induced by down-regulation of Bcl-xL. In all three cell lines caspase activation following antisense treatment was associated with nuclear condensation and fragmentation, another hallmark of apoptosis (insets in Fig. 6Citation ).



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Fig. 6. Caspase-3-like protease activity in lung cancer cells after treatment with antisense oligonucleotides. SW2, NCI-H125, or A549 cells were treated without (Untreated, Lipofectin) or with 600 nM oligonucleotides, and caspase-3-like protease activity was measured by use of a colorimetric assay 40 and 64 h after the start of treatment. Representative results of one of three independent experiments are shown. Inset, Hoechst staining of cells 40 h after treatment with oligonucleotide 4625 reveals nuclear condensation and fragmentation (original magnification, x1000).

 

    DISCUSSION
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated levels of Bcl-2 and Bcl-xL are frequently found in tumors where they may exert nonredundant and distinct biological roles in cell survival, tumor development, and drug resistance (3 , 4) . The complexity of the functional and regulatory interrelationship between Bcl-2 and Bcl-xL is demonstrated by the findings that tumor cells can switch expression from Bcl-2 to Bcl-xL (5) and that ectopic overexpression of Bcl-2 can result in the reciprocal down-regulation of Bcl-xL (20) . In vivo, the situation is even more complicated because most solid tumors are genetically and phenotypically heterogeneous cell populations in which both antiapoptotic proteins potentially contribute to cell survival. Most small cell lung cancer cells express both Bcl-2 and Bcl-xL, whereas in non-small cell lung cancer cells Bcl-xL is clearly more prevalent and likely to be a more critical repressor of apoptosis than Bcl-2 (21) . In clinical practice, lung cancer may exhibit more than one histological pattern (22) , and there is evidence that even transitions from a small cell to a non-small cell phenotype may occur (23) . This level of heterogeneity makes it difficult to predict which of the antiapoptotic proteins might be biologically more critical for cell survival and which might be the more relevant molecular target for antisense therapy.

The potency of an antisense oligonucleotide to inhibit the expression of two different genes simultaneously depends on the availability of complementary target sequences with a high degree of homology between the two mRNA species. Bcl-2 and bcl-x share a number of such homologous sequences. By use of sequence alignment (12) , we have identified a region comprising nucleotides 605–624 and 687–706 of the bcl-2 and bcl-xL mRNA, respectively, which represents a particularly interesting site for antisense hybridization because it: (a) differs by only three nucleotides (Fig. 1)Citation ; and (b) is located at the splice junction site of bcl-x and neither occurs in the proapoptotic splice variant bcl-xS nor the other proapoptotic bcl-2 family members such as bax and bak.

A major challenge for the design of antisense oligonucleotides is the apparent paucity of mRNA sites that can be targeted efficiently, and several in vitro techniques have been proposed to test the accessibility of oligonucleotides to complementary regions in mRNA (11) . We have used the RNAdraw computer program (13) to unveil that in both mRNA species our sequence of interest presents in the form of two small single-stranded loops separated by a short base-paired spacer. Such a structural motif is typically found in the translation initiation regions of many mRNAs and could prove to be an accessible structure for antisense oligonucleotides targeting the bcl-2 coding region (24) .

Many of the problems related to antisense therapy, including oligonucleotide stability, affinity, or delivery, are gradually being overcome through advances in oligonucleotide chemistry, such as the introduction of a phosphorothioate backbone and/or 2' modifications of the ribose moieties (6 , 14 , 25) . Because these modifications, however, abrogate the ability of oligonucleotides to activate RNase H, "gapmers" with only few nucleotides at the 5' and 3' end being modified have been used (16) . A major advantage of MOE-modified oligonucleotides is their increased hybridization affinity and resistance to nucleases (14 , 16) . These improved functional properties may also explain the relatively low degree of unspecific nonantisense-related toxicity of these second generation analogues against target cells compared with their first generation "deoxy" counterparts (data not shown). In antisense 4625, MOE modifications were made to selected riboses at the 5' and 3' end, including two of the three mismatching nucleotides of the bcl-2/bcl-xL sequences. This resulted in a potent bcl-2/bcl-xL bispecific compound, as demonstrated by real-time PCR and Western blot analysis. Down-regulation of Bcl-xL and Bcl-2 also occurred in the presence of the caspase inhibitor zVAD.fmk, ruling out the possibility that the onset of apoptosis as induced by the antisense oligonucleotides caused caspase-mediated degradation of these proteins (17 , 18) .

Small cell lung cancer cells, including the cell line SW2, overexpress Bcl-2 and Bcl-xL, whereas most non-small cell lung cancer cells, including the cell lines NCI-H125 and A549, preferentially express Bcl-xL (21 , 26) . Our previous studies unveiled Bcl-2 as a survival factor for small cell lung cancer cells, the down-regulation of which facilitates apoptosis and sensitizes to chemotherapy (7 , 27) . Here, we show that in SW2 cells apoptosis was most strongly induced by the oligonucleotides 4625 and 4627, which both efficiently inhibited the expression of Bcl-2 and Bcl-xL. Although Bcl-xL was significantly expressed in SW2 cells, its down-regulation alone by use of the preferentially bcl-xL-specific oligonucleotide 4259 did not induce apoptosis, suggesting this death antagonist to be a less critical survival factor for these tumor cells. This is in contrast to other solid tumors, including non-small cell lung cancer, where Bcl-xL was found to be a positive modulator of drug resistance (21 , 28 , 29) . In agreement with this finding, apoptosis was induced in the two non-small cell lung cancer cell lines NCI-H125 and A549 after treatment with oligonucleotide 4259, indicating that overexpression of Bcl-xL correlated with its importance as a survival factor in these cells.

The correlation between the increase in caspase activity and the number of dead cells measured by PI uptake was not linear, and the 100-fold increase in caspase activity in A549 cells resulted in an apoptotic rate of 33%, which was only slightly more than observed in the other two cell lines. Morphological manifestations of apoptosis, such as nuclear fragmentation detected by Hoechst staining or loss of plasma membrane integrity and PI uptake, however, are of short duration and relatively late events during apoptosis. FACS analysis of antisense-treated cells revealed a large amount of cell debris, indicating that at the time of analysis a substantial fraction of dead cells was, indeed, already disintegrated and no longer detectable.

Recently, Ackermann et al. (8) reported the apoptosis-inducing effect of another MOE-modified bcl-xL antisense oligonucleotide on umbilical vein endothelial cells. This compound, however, targets a region located 100 nucleotides upstream of our target sequence, which also occurs in the mRNA of the proapoptotic splice variant bcl-xS. Whether this represents a potential drawback for therapeutic use may depend on the tumor target. As shown for other lung cancer cell lines (21) , low levels of Bcl-xS were also detectable in SW2 cells (data not shown), and up-regulation of Bcl-xS was described in other neuroendocrine tumor cells undergoing apoptosis (30) .

The present study describes the design and functional evaluation of novel MOE-modified phosphorothioate antisense oligonucleotides with bispecificity for bcl-2 and bcl-xL. Our data suggest that the use of oligonucleotide 4625, which most efficiently inhibited bcl-2 and bcl-xL expression and induced apoptosis in lung cancer cells, deserves attention as a novel approach to cancer therapy.


    FOOTNOTES
 
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.

1 Supported by Grant 31-40473.94 from the Swiss National Science Foundation, Grant 549-9-1997 from the Krebsforschung Schweiz, and the Stiftung zum Baugarten (Zürich, Switzerland). Back

2 To whom requests for reprints should be addressed, at Division of Oncology, Department of Internal Medicine, University Hospital Zürich, Haeldeliweg 4, CH-8044 Zürich, Switzerland. Phone: 0041-1-6342877; Fax: 0041-1-6342872. Back

3 The abbreviations used are: MOE, O-methoxy-ethoxy; FACS, fluorescence-activated cell sorting; PI, propidium iodide. Back

Received 10/ 7/99; revised 2/28/00; accepted 3/ 2/00.


    REFERENCES
 Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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