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Experimental Therapeutics, Preclinical Pharmacology |
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.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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'-
-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).
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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 1
. 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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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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, 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)
. 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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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. 2
and 3
and again
suggest oligonucleotide 4625 as the most potent bcl-2/bcl-xL
bispecific antisense compound.
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, 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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) 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. 6
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. 6
).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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)
; 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.
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FOOTNOTES |
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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). 
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.
3 The abbreviations used are: MOE,
O-methoxy-ethoxy; FACS, fluorescence-activated cell
sorting; PI, propidium iodide.
Received 10/ 7/99; revised 2/28/00; accepted 3/ 2/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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G. Tortora, R. Caputo, V. Damiano, R. Caputo, T. Troiani, B. M. Veneziani, S. De Placido, A. R. Bianco, U. Zangemeister-Wittke, and F. Ciardiello Combined Targeted Inhibition of bcl-2, bcl-XL, Epidermal Growth Factor Receptor, and Protein Kinase A Type I Causes Potent Antitumor, Apoptotic, and Antiangiogenic Activity Clin. Cancer Res., February 1, 2003; 9(2): 866 - 871. [Abstract] [Full Text] [PDF]
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 B. Jahrsdorfer, R. Jox, L. Muhlenhoff, K. Tschoep, A. Krug, S. Rothenfusser, G. Meinhardt, B. Emmerich, S. Endres, and G. Hartmann Modulation of malignant B cell activation and apoptosis by bcl-2 antisense ODN and immunostimulatory CpG ODN J. Leukoc. Biol., July 1, 2002; 72(1): 83 - 92. [Abstract] [Full Text] [PDF]
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 W. R. Smythe, I. Mohuiddin, M. Ozveran, and X. X. Cao Antisense therapy for malignant mesothelioma with oligonucleotides targeting the bcl-xl gene product J. Thorac. Cardiovasc. Surg., June 1, 2002; 123(6): 1191 - 1198. [Abstract] [Full Text] [PDF]
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O. E. Pardo, A. Arcaro, G. Salerno, S. Raguz, J. Downward, and M. J. Seckl Fibroblast Growth Factor-2 Induces Translational Regulation of Bcl-XL and Bcl-2 via a MEK-dependent Pathway. CORRELATION WITH RESISTANCE TO ETOPOSIDE-INDUCED APOPTOSIS J. Biol. Chem., March 29, 2002; 277(14): 12040 - 12046. [Abstract] [Full Text] [PDF]
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 A. S. Jun, S. H. Liu, E. H. Koo, D. V. Do, W. J. Stark, and J. D. Gottsch Microarray Analysis of Gene Expression in Human Donor Corneas Arch Ophthalmol, November 1, 2001; 119(11): 1629 - 1634. [Abstract] [Full Text] [PDF]
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M. S. Rieber, U. Zangemeister-Wittke, and M. Rieber p53-independent Induction of Apoptosis in Human Melanoma Cells by a bcl-2/bcl-xL Bispecific Antisense Oligonucleotide Clin. Cancer Res., May 1, 2001; 7(5): 1446 - 1451. [Abstract] [Full Text]
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 O. Gautschi, S. Tschopp, R. A. Olie, S. H. Leech, A. P. Simoes-Wust, A. Ziegler, B. Baumann, B. Odermatt, J. Hall, R. A. Stahel, et al. Activity of a Novel bcl-2/bcl-xL-Bispecific Antisense Oligonucleotide Against Tumors of Diverse Histologic Origins J Natl Cancer Inst, March 21, 2001; 93(6): 463 - 471. [Abstract] [Full Text] [PDF]
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I. Lebedeva, R. Rando, J. Ojwang, P. Cossum, and C. A. Stein Bcl-xL in Prostate Cancer Cells: Effects of Overexpression and Down-Regulation on Chemosensitivity Cancer Res., November 1, 2000; 60(21): 6052 - 6060. [Abstract] [Full Text]
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