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Experimental Therapeutics |
Departments of Medicine [I. L., C. A. S.] and Pharmacology [C. A. S.], Columbia University, New York, New York 10032, and Aronex Pharmaceuticals, The Woodlands, Texas 77381-1191 [R. R., J. O., P. C.]
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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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Bcl-x is a relatively new member of the Bcl-2 family of apoptosis-related proteins, and its expression has been detected in a range of normal tissues, particularly in the central nervous system and thymus. Immunocytochemical studies have demonstrated that the Bcl-x protein can be detected in numerous tumor cell lines as well. Expression has been found in the epithelial cells of the normal prostate (1) , where it is speculated that it contributes to the hormone-dependent control of programmed cell death. Subsequently, Krajewski et al. (2) found that 64 of 64 (100%) cases of adenocarcinoma of the prostate stained positively for Bcl-x protein. Staining intensity seemed to be correlated with increasing Gleason score and the presence of metastases. Because Bcl-xS, a proapoptotic protein, appeared not to be expressed either in prostatic tumors or in prostate cell lines in tissue culture, this staining most likely originated solely from the Bcl-xL protein. Similarly, Bcl-2 expression is also found quite extensively in primary prostate cancer specimens (3) , although it has been observed in only 33% of samples of prostate tumors obtained from the bone marrow of patients with hormone-refractory disease (4) . At the present time, it appears that although Bcl-xL and Bcl-2 may have similar antiapoptotic functions, it is not clear why both are co-expressed in at least some prostate cancer cells, although it is possible that overexpression of Bcl-xL and Bcl-2 protein might be factors enabling prostate cancer cells to survive in an androgen-deprived environment (5) . Forced overexpression of Bcl-2 has been used in many experiments to demonstrate chemodesensitization of prostate cancer cells (5, 6, 7) . However, it is unclear whether the function of Bcl-2 at forcibly overexpressed intracellular levels bears a resemblance to its basal function.
Both Bcl-2 and Bcl-xL may prevent apoptosis by similar mechanisms. Bcl-xL (like Bcl-2) clearly acts to regulate mitochondrial membrane potential and volume (8) and can block the apoptosis-inducing release of cytochrome c and apoptosis inhibitory factor into the cytoplasm (8, 9, 10, 11, 12, 13) . In addition, Bcl-2 and Bcl-xL also may suppress apoptosis in a cytochrome c-independent manner (14) , perhaps because of their ability to inhibit cytotoxin-induced caspase-3 activity and subsequent poly(ADP-ribose) polymerase cleavage and lamin B1 degradation (15 , 16) .
The Mr 25,600 Bcl-xL protein has been shown in a number of cell lines to be a potent protector of cellular apoptosis induced by antineoplastic agents (9) . When transferred into the murine interleukin 3-dependent prolymphocytic line FL5.12, Bcl-xL greatly reduced the proapoptotic effects of bleomycin, cisplatin, etoposide, vincristine, and doxorubicin (17) . In U937 cells, inhibitory effects on camptothecin-induced apoptosis were shown to be dependent on the intracellular concentration of Bcl-xL protein. Diminution of cell death in response to etoposide, vinblastine, paclitaxel, and cisplatin were also observed (18 , 19) . Bcl-xL can also block cellular apoptosis in settings in which Bcl-2 is ineffectual (20) . Furthermore, several agents, such as butyrate in human fibroblasts (21) and paclitaxel in LNCaP and PC-3 human prostate cancer cells (22) , induce apoptosis that can be correlated with substantial decreases in the expression of Bcl-xL protein, and Bcl-xL expression, but not Bcl-2 or Bax expression, has been shown to decrease prior to the onset of apoptosis in CTLL-2 cells (23 , 24) . Finally, intracellular cleavage of Bcl-xL by caspase-3-like proteases has been associated with increased rates of cell death (23) .
In this work, we demonstrate that an increase in Bcl-xL expression in LNCaP and PC-3 prostate cancer cells desensitizes them to the effects of cytotoxic chemotherapy. We used the directed oligonucleotide ("antisense") biotechnology to down-regulate the expression of Bcl-xL in these cells. Our results demonstrate that the sensitivity of prostate cancer cells to a wide variety of cytotoxic agents can be markedly increased by this strategy. On the basis of the use of two antisense strategies, this report suggests that basal Bcl-2 protein expression may not play a major role in the antiapoptotic phenotype in prostate cancer cell lines when treated with cytotoxic agents.
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MATERIALS AND METHODS |
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Cell Culture and Transformation of Cell Lines with Bcl-xL
Plasmid.
Human PC-3 and LNCaP prostate cancer cell lines were obtained from
American Type Culture Collection (Rockville, MD). They were grown in
RPMI 1640 (Life Technologies, Inc., Grand Island, NY), containing 10%
(v/v) heat inactivated (56°C) fetal bovine serum
FBS2
(Life Technologies, Inc., Grand Island, NY), supplemented by 1%
nonessential amino acids, 1% pyruvate, 100 units/ml penicillin G
sodium, and 100 µg/ml streptomycin sulfate. All cell lines were
cultured in 5% CO2 atmosphere and were routinely
passaged when 90–95% confluent.
LNCaP cells were transfected with the neomycin-selectable pSFFV/Bcl-xL plasmid (obtained from Dr. Stanley Korsmeyer, Washington University School of Medicine, St. Louis, MO) or with a control, neomycin-resistant expression vector pSFFV. Aliquots containing 10 µg of plasmid and 5 µg of Lipofectin reagent (Life Technologies, Inc., Gaithersburg, MD) in serum-free OPTI-MEM (Life Technologies, Inc.) were added to cultured cells. The transfection medium was replaced 4 h later with RPMI-10% FBS medium. Individual colonies were selected from these plates after approximately 3–4 weeks of routine maintenance in RPMI-10% FBS containing 0.6 mg/ml G418 sulfate (Geneticin; Life Technologies, Inc.). The Bcl-xL-transformed LNCaP cells were cultured in RPMI-10% FBS, supplemented with 0.3 mg/ml G418 sulfate in 5% CO2 atmosphere.
Oligonucleotides.
In the present study, we have used a directed oligonucleotide
("antisense") strategy to down-regulate Bcl-xL protein expression
in prostate cancer cell lines. To maximize efficacy, we initially
screened 20 different oligonucleotide sequences (10 20-mers and 10
18-mers) complementary to the Bcl-xL mRNA. Because at the present time
no computer algorithm has sufficient predictive power of activity,
sequences were randomly chosen and are shown in Fig. 1
. To reduce nonsequence specificity, phosphorothioate linkages were only
placed 5' to each pyrimidine. Phosphodiester linkages were retained 5'
to each purine, because in this setting they are almost as nuclease
resistant as a phosphorothioate linkage (25
, 26)
.
Isosequential oligonucleotides were also synthesized with the
C5-propyne modification. Oligonucleotides in Fig. 1
with even numbers
have unmodified heterocyclic bases, whereas those with odd numbers
contained a C-propynyl modification at each pyrimidine.
Oligonucleotides were screened for their antisense activity to
down-regulate Bcl-xL protein expression using Western blot analysis.
All oligonucleotides were prepared and purified as described previously
(27)
and were at least 95% full-length when analyzed by
reverse-phase high-pressure liquid chromatography.
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Oligonucleotides were delivered to the cells complexed with the cationic porphyrins TMP and TAP (PC-3 and LNCaP cells, respectively). Oligonucleotide was premixed with TAP or TMP in OPTI-MEM medium to the final concentrations described in "Results" for each cell line. A complex was formed for 15 min at room temperature; cells were washed once with OPTI-MEM and treated with prepared complex for 5 h at 37°C. Then cells were washed once with RPMI-10% FBS and were allowed to recover in RPMI-10% FBS for another 20 h. At this time, cells were washed twice with PBS, and then protein and/or mRNA were extracted as described and analyzed.
Western Blot Analysis.
Cells treated with oligonucleotide-porphyrin complex were washed twice
with cold PBS and lysed on ice for 30 min in 100 µl of cold RIPA
buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl,
0.1% SDS, 1% NP40, and 0.5% sodium deoxycholate] with freshly added
0.1 mg/ml phenylmethylsulfonyl fluoride, 1 mM sodium
orthovanadate, and 1 mg/ml aprotinin. Cell debris was removed by
centrifugation at 14,000 x g for 10 min at
4°C. Protein concentrations were determined using the Bio-Rad protein
assay system (Bio-Rad Laboratories, Richmond, CA).
Aliquots of cell extracts containing 20–50 mg of total protein were resolved in 12% SDS-PAGE and transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA). Filters were blocked for 1 h at room temperature in Blotto A [5% nonfat milk powder in TBS-T: 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.05% Tween 20] and then incubated for 1 h at room temperature in Blotto A containing a 1:200 dilution of either rabbit anti-Bcl-xL, anti-Bax, or anti-Bak polyclonal antibodies (all from Santa Cruz Biotechnology Inc., Santa Cruz, CA). After washing in TBS-T buffer (three times for 5 min each time, at room temperature), filters were incubated for 45 min at room temperature in Blotto A containing a 1:10,000 dilution of corresponding peroxidase-conjugated, antirabbit secondary antibody (Amersham, Arlington Heights, IL). To detect Bcl-2, a 1:500 dilution of an anti-Bcl-2 mouse mAb (Dako, Carpinteria, CA) was added in 1% BSA/TBS. After washing in TBS-T buffer (three times for 5 min each time, at room temperature), filters were incubated for 45 min at room temperature in 1% BSA/TBS buffer containing a 1:3,000 dilution of peroxidase-conjugated, antimouse secondary antibody (Amersham). After washing in TBS-T, enhanced chemiluminescence was performed according the recommendation of the manufacturer. Bcl-xL protein expression, as determined by Western blot analysis, was quantitatively analyzed via laser-scanning densitometry using NIH Image Version 1.61 software. All results were calculated as a percentage of Bcl-xL protein expression in treated versus untreated cells.
RNA Isolation and Northern Blot Analysis.
Total RNA was isolated from 5 x 107 cells using an RNeasy Mini kit from Qiagen
(Santa Clarita, CA). Forty µg of total RNA were electrophoresed on a
1.2% denaturing agarose gel containing formaldehyde, transferred to
Nytran-Plus nylon membranes (Schleicher & Schuell, Keene, NH), and
covalently bound to a membrane by UV irradiation. The 0.5-kbp GAPDH
probe was PCR generated. The Bcl-xL (0.7-kbp) and Bcl-2 (0.44-kbp) cDNA
fragments generated, respectively, from the EcoRI-restricted
pSFFV/Bcl-xL and the HindIII/SstI pcDNA3/Bcl-2
(5)
plasmids, and 32P-labeled
by the random primer method, were hybridized to the filter-bound RNA in
ULTRAHyb solution (Ambion, Inc., Austin, TX) overnight at 42°C. The
membrane was washed two times for 15 min in 1x SSC, 0.1% SDS at
42°C and two times for 10 min in 0.1X SSC, 0.1% SDS at 42°C. The
filter was exposed to Kodak X-ray film with intensifying screens for 3
days at -70°C and developed.
MTT Assay for Determination of Cellular Viability.
The cytotoxicity of the combinations of drugs with oligonucleotides was
determined by measurement of cell viability by use of the MTT assay.
LNCaP or PC-3 cells were seeded in 96-well tissue culture plates and
treated the next day with oligonucleotides and drugs at the indicated
concentrations as described in "Results." Drug-containing medium
was then removed, and the cells were washed once with PBS. RPMI/10%
FCS containing 0.5 mg/ml MTT (Sigma) was added to each well. The cells
were incubated at 37°C for 4 h and then an equal amount of
solubilization solution (0.04 N HCl in isopropyl alcohol)
was added to each well and mixed thoroughly to dissolve the crystals of
MTT formazan. After all of the crystals were dissolved, the plates were
read on a Dynatech MR600 Microplate Reader at 540 nm. Statistical
analysis of the results of the MTT experiments was performed using the
Analysis ToolPack provided by Microsoft Excel. A Student two-sample
t test, assuming unequal variances, was used to determine
the equality of the means of two samples. The confidence level (
)
was 0.05.
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RESULTS |
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A, the LNCaP-Bcl-xL line expresses 
15-fold more Bcl-xL
protein than does the empty-vector LNCaP/neo. Similarly, PC-3-Bcl-xL
cells express 4–5-fold more Bcl-xL protein than PC-3/neo cells. In
neither cell line were levels of Bcl-2 or Bax proteins altered (data
not shown). The effects of Bcl-xL overexpression on the sensitivity of
these cell lines to cytotoxic chemotherapeutic agents is shown in Fig. 2B
. MTT determinations were performed after 48 h of
continuous treatment with the cytotoxic agents. Under these conditions,
cellular viability after treatment with either paclitaxel or
mitoxantrone (chosen because both are clinically active) is decreased
by approximately 40–50% in the overexpressing cell line
versus either the wild-type cells or the LNCaP/neo cells.
This decrease is statistically significant (P < 10-3). It should be noted that at
concentrations of paclitaxel >100 nM,
Bcl-xL-dependent chemosensitization was no longer observed because the
drug was equally toxic to the overexpressing, neo-transfected, and
wild-type cells.
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; MTT assays were
performed after 72 h of continuous drug treatment). Taken
together, these data strongly suggest that Bcl-xL impacts greatly on
the chemosensitivity of prostate cancer cells in tissue culture.
Down-Regulation of Bcl-xL Expression by Chimeric, C5-Propynylated
Oligonucleotides.
Because of unacceptable cytotoxicity, we could not use cationic lipids,
such as Lipofectin, as delivery agents in prostate cancer cells.
Instead, we used two cationic porphyrins, TMP and TAP, to deliver the
oligonucleotides (27)
. For unclear reasons, however, the
porphyrin carrier required for maximal antisense activity was prostate
cancer cell-line dependent. For LNCaP cells, only TAP but not TMP was
effective, whereas the converse was true for PC-3 cells, where only TMP
was active. For LNCaP cells, optimally active complexes were formed at
concentrations of 3 µM TAP/1 µM
oligonucleotide; for PC-3 cells, 7 µM TMP/2
µM oligonucleotide was required.
Down-regulation of the expression of Bcl-xL protein by C5-propynylated
chimeric oligonucleotides was demonstrated by Western blotting, as
shown in Fig. 3A
. (A Mr 19,000 band that
would correspond to the Bcl-xS protein could not be detected in either
of these cell lines.) Equal loading of protein samples was achieved by
measurement of protein concentrations by the Bradford assay (Bio-Rad
Laboratories). In some cases, equal protein loading was verified by
examination of actin expression. These results correlated exactly with
protein concentration determination. In LNCaP cells, the most active
sequences were 29, 41, 57, and 61. Other
oligomers presented in Fig. 3
A, such as 33, 45,
38, and 53 presented here, showed little or no
activity. The full spectrum of oligonucleotide activity (or lack
thereof) is shown in Fig. 3B
(LNCaP cells) and Fig. 3C
(PC-3 cells). In PC-3 cells, optimal results were
obtained with 41, and 29 and 61 were
also active. In general, oligomers not containing C5-propynylated
pyrimidine residues were not as active as the substituted molecules.
Compared with the untreated cells or cells treated with inactive
oligonucleotides, the most active oligonucleotides (Figs. 4
and 5)
consistently decreased the level of Bcl-xL expression to 5–30% of
the control level. Oligonucleotide treatment also did not cause any
reduction in the total amount of cellular protein, nor, as demonstrated
by MTT assay, did it inhibit cellular growth.
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, along with the corresponding Northern blots.
Down-regulation of bcl-x mRNA levels occurs congruently with
down-regulation of Bcl-xL protein expression, thus supporting an RNase
H-based mechanism of action. In LNCaP, 41 is perhaps the
most active oligomer, with both 29 and 41 being
highly active in PC-3 cells.
Antisense Bcl-xL Oligonucleotides Also Down-Regulate Bcl-2 but not
Bax or Bak.
The ability of cell to undergo apoptosis is determined by the
ratio between anti- and proapoptotic members of the Bcl-2 family
(28)
. We therefore investigated whether treatment of cells
with antisense Bcl-xL oligonucleotides affects these Bcl-xL-related
proteins (Fig. 6)
. Oligonucleotides 29, 41, and 43, when delivered
with TMP to PC-3 cells, caused down-regulation of both antiapoptotic
proteins Bcl-xL and Bcl-2 but appeared to up-regulate Bax protein
expression. Bak protein expression, however, was not changed after
treatment. In LNCaP cells, however, the diminution in Bcl-2 expression
after treatment with 41 was not as great as in PC-3 cells,
but neither Bax nor Bak protein expression was affected. Table 1
presents the results on Bcl-xL, Bcl-2, and Bax protein expression after
treatment of LNCaP and PC-3 cells with the most active C5-propynylated
oligonucleotides. It should be noted that in several instances
(e.g., with oligomers 57 and 61),
although Bcl-xL and Bcl-2 expression decreased, Bax expression also
decreased. Additional evidence for the co-down-regulation of Bcl-2 and
Bcl-xL in LNCaP and PC-3 cells by 41 is provided in Fig. 6
.
Western blots for Bcl-2 and Bcl-xL demonstrate the previously
demonstrated down-regulation by 41; a Northern blot
demonstrates that Bcl-2 mRNA expression is down-regulated as well,
whereas the expression of GAPDH mRNA is unchanged.
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, after 3 days of drug treatment which followed 5 h of antisense
oligonucleotide treatment, cell viability was significantly decreased,
as determined by MTT assay. (It should be kept in mind that the active
oligonucleotides also down-regulate Bcl-2 expression.) For the most
active oligomer, 41, MTT expression (compared with drug-only
treated cells; for all experiments, n = 5) in
the presence of 0.1 µM mitoxantrone was
decreased 39.8 ± 4.3% (P < 10-10); in the presence of 25
nM paclitaxel, 34 ± 6.2%
(P < 10-4); in the
presence of 50 nM Taxotere, by 33.1 ± 4.8% (P < 10-5); in the presence of 10
µM carboplatin, by 47 ± 7.2%
(P < 10-7); and by
49.9 ± 2.0% in the presence of etoposide
(P < 10-3). Oligomers
57 and 61, also highly statistically significant
(P < 10-3), increased
sensitivity to these cytotoxic agents but did not tend to be as active
as 41, perhaps because these two also inhibit expression of
Bax protein (Table 1)
. However, neither the C5-propynylated controls
45 and 47 nor the non-C5-propynylated species
34, 38, 46, or 62 produced any
significant change in cellular viability.
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. As shown in Fig. 8
, these gap-mers down-regulated the expression of Bcl-xL protein
(41m and 57m are the most active). Bcl-2
expression, as expected, was not dramatically decreased, and Bax
expression was unchanged after treatment. The four-base mutant
oligomers did not down-regulate either Bcl-xL or Bcl-2 expression.
However, Northern analysis did not demonstrate any decrease in the
expression of Bcl-xL mRNA, even by the most active oligomers (Fig. 9)
. It is thus very unlikely that the down-regulation of Bcl-xL protein
expression by 41m is RNase H dependent. Nevertheless,
treatment of both LNCaP and PC-3 cells with the most active oligomers
(41m for LNCAP and 41m or 57m for PC-3
cells) led to significant chemosensitization (P < 10-4) to a large number of cytotoxic
agents when compared with cells treated only with drugs (Fig. 10)
. These agents included docetaxel (25 nM;
25.4 ± 2.9%, P < 10-5), paclitaxel (25 nM;
46.9 ± 4.3%, P < 10-4), vinblastine (1
µM; 70.6 ± 4.6%,
P < 10-5), etoposide
(10 µM; 47.2 ± 1.9%,
P < 10-3), and
carboplatin (10 µM; 47.9 ± 11.3%, P = 10-4) in
PC-3 cells. 41mut and 57mut, which contain four
mutated bases, were ineffective (i.e., identical to
control). Similar, statistically significant (P < 10-4) sensitization to taxanes, velban
and mitoxantrone, were also observed in LNCaP cells with 41m
(10 nM paclitaxel, 33.5 ± 4.4%;
20 nM docetaxel, 43.7 ± 5.7%; 1
µM mitoxantrone, 49.7 ± 4.2%;
1 µM vinblastine, 46.5 ± 5.4%), whereas 41mut was ineffective. In contrast, PC-3
cells were not sensitized to the cytotoxic effects of
all-trans-retinoic acid after treatment with 41m.
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DISCUSSION |
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Both LNCaP and PC-3 cells endogenously express Bcl-2 protein (29
, 30)
. Although Bcl-2 and Bcl-xL appear to "repress a common
pathway of cell death" (31)
and in certain circumstances
appear to be functionally equivalent (32
, 33)
, some
evidence indicates that under other circumstances, their sites of
action may be distinct. For example, in clones of FL5.12 lymphoid cells
containing approximately equal amounts of both Bcl-2 and Bcl-xL
protein, the latter provided significantly greater protection against
etoposide and teniposide, and against three S-phase agents including
methotrexate, 5-fluorouracil, and hydroxyurea (34)
. In
WEHI-231.7 JM lymphoid cells overexpressing Bcl-xL, the apoptotic
response to cyclosporin A, FK-506, and rapamycin was blocked
(20)
. In contrast, apoptosis was not blocked when this
line was engineered to overexpress Bcl-2. In addition, Bcl-xL, but not
Bcl-2, could protect MCF-7 cells by preventing ceramide accumulation
induced either by tumor necrosis factor- or camptothecin
(35)
.
Previous experiments have also demonstrated that down-regulation of Bcl-xL expression can result in chemosensitization in at least some cell types. 20-mer antisense phosphorothioate oligonucleotides (0.45 µM) delivered by the lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate to eosinophils blocked granulocyte/macrophage-colony stimulating factor or interleukin 5-induced Bcl-xL up-regulation and inhibited the prevention of eosinophil death by 34% (36) . Taylor et al. (37) used 20-mer "gap-mers" to down-regulate Bcl-xL expression in human neonatal keratinocytes and in the A549 lung epithelial tumor cell line. Consistent with our results, they demonstrated that only approximately 1–8% of the cells died after treatment; however, with subsequent treatment by UV-B radiation or cisplatin, the number of apoptotic cells increased by 2–3-fold over the control, mismatched oligomer-treated cells. In subsequent experiments, Ackermann et al. (38) demonstrated that 10–25% of treated human umbilical vein endothelial cells underwent apoptosis after oligonucleotide-induced down-regulation of Bcl-xL protein expression. The sensitivity of the cells to staurosporine and ceramide were also increased, which is an interesting observation in light of the data of El-Assaad et al. (35) that Bcl-xL does not appear to protect MCF-7 breast cancer cells from ceramide-induced apoptosis. The lack of significant apoptosis in prostate cancer cells in which Bcl-xL (and Bcl-2) expression has been dramatically reduced indicates that these are not critical proteins for basal viability of these cells.
It has been demonstrated (37, 38, 39) that the best way at this time to find active antisense oligonucleotide sequences is to manually divide the targeted mRNA sequence into a series of 18–20-mers and target each of these fragments individually. Subsequently, to maximize the yield of active oligonucleotides, we improved the interaction (i.e., raised the Tm) between the oligomer and the target by using a C5-propyne substituent at each pyrimidine moiety (40, 41, 42) . This modification promotes oligonucleotide base stacking (42) , which appears to favor duplex formation. As expected, in the absence of this modification, the extent of antisense down-regulation decreased even for the most active species. Furthermore, to decrease the nonsequence specificity, which is such a prominent feature of the behavior of phosphorothioate linkages (43 , 44) , we retained phosphodiester linkages 5' to each purine residue, at which location they are resistant to nucleases (25 , 26) , as opposed to those 5' to pyrimidine, which are highly sensitive. However, the C5-propyne-modified all-phosphorothioate 41 down-regulated Bcl-xL protein expression with identical efficacy as the chimeric 41. Because the effects on Bcl-xL protein expression (not shown) of the more nonspecific all-phosphorothioate C5-propynylated oligomers were identical to those of the chimerics, which have reduced phosphorothioate content, it seems logical to assume that the effects of the chimerics were not solely attributable to nonspecificity.
Treatment of either LNCaP or PC-3 cells with porphyrin (TAP for LNCaP and TMP for PC-3 cells) complexes of the active C5-propyne chimeric oligonucleotides led to co-down-regulation of Bcl-xL and Bcl-2 protein expression but did not lead to significant (>10%) spontaneous cellular apoptosis. This contrasts with the apoptosis observed in diverse cell types (including prostate cancer cell lines) with G3139 (45) , an all-phosphorothioate 18-mer targeted to the initiation codon region of Bcl-2. In light of our observations, the strongly proapoptotic effects of G3139 indicate that down-regulation of Bcl-2 protein expression by this molecule may be necessary, but is not sufficient, for apoptosis initiation; an additional, perhaps necessary toxic insult may be provided by the lipid carrier (Lipofectin). An additional possibility is that G3139 can down-regulate the expression of perhaps many additional genes via the process of "irrelevant cleavage" (46) . Because of the "low stringency," nonselective mechanism of action of RNase H, sequence homology of the antisense oligonucleotide with nontargeted mRNAs may lead to their cleavage. Indeed, this process is probably responsible for the co-down-regulation of Bcl-xL and Bcl-2 by the C-5-propynylated chimeric oligomers presented in this work. In the study presented here, the GAGTC five-base motif found in the antisense Bcl-xL oligomer 41 is also found at both 3290–3294 and 4554–4558 nucleotides in the Bcl-2 mRNA. Similarly, oligomers 29 and 57, which down-regulate both Bcl-xL and Bcl-2 in PC-3 cells, have a region of seven-base homology (TGGTTGA for 29 and GGAGATG for 57) at nucleotides 2532–2538 and 524–530, respectively. Of course, for any oligonucleotide, partially or fully complementary, to elicit RNase H activity it must hybridize to its complement; and it is also true that many if not most complementary sequences are either not accessible to sites on the target because of mRNA secondary structure and folding, or even if accessible, may not form sufficiently stable complexes to be recognized and cleaved by RNase H (47) . Interestingly, the antisense Bcl-2 oligomer G3139 has a single seven-base (and several five-base) regions of contiguous homology with the Bcl-x mRNA, sufficient overlap for RNase H competency (48) . Furthermore, preliminary results obtained from the treatment of RCC-E kidney cancer cells by G3139 demonstrate co-down-regulation of Bcl-2 and Bcl-xL protein expression.3
Down-regulation of Bcl-xL and Bcl-2 protein expression by a single oligonucleotide, achieved by taking advantage of the "low stringency" requirements of RNase H, has been accomplished recently (49) . Such a bispecific oligonucleotide induced apoptosis in the SW2 small cell lung cancer cell line, where Bcl-2 has been shown to be a significant antiapoptotic effector. However, in other cell lines, where Bcl-xL was found to be the important antiapoptotic effector, the bispecific antisense oligonucleotide was not a more efficient inducer of apoptosis than was an antisense oligonucleotide that down-regulates Bcl-xL expression only. These results correlate exactly with our own. However, the use of RNase H-competent oligonucleotides to co-down-regulate both Bcl-2 and Bcl-xL may affect the expression of other, as yet unknown, genes because of irrelevant cleavage. It is entirely possible that these unintended effects may also contribute to the observed chemosensitization.
A reduction in RNase H activity, and thus in irrelevant cleavage, can
be obtained by eliminating RNase H-competent deoxyribose linkages at
the molecular termini and substituting them with non-RNase H-competent
2'-O-methyl "gap-mers." In this work, as in others
(50
, 51)
, in such "gap-mers" a central region of
phosphorothioate oligonucleotide was included to retain some, albeit
reduced, RNase H activity. As predicted, perhaps in part because of
reduced irrelevant cleavage, neither 41m nor 57m
down-regulated the expression of Bcl-2 protein. However, despite the
ability of these oligonucleotides to down-regulate Bcl-xL protein
expression, Northern analysis (Fig. 9)
did not demonstrate any decline
in Bcl-xL mRNA expression, demonstrating that this decrease in protein
expression was non-RNase H dependent. Nevertheless, the fact that this
down-regulation occurred in the absence of a diminution in mRNA
expression is surprising in light of the activity of the 80S ribosome
(52
, 53)
, which in theory should have unwound the
mRNA-"gap-mer" oligonucleotide duplex. In addition, the eight-base
core phosphorothioate region should certainly have been sufficient for
RNase H competency. Therefore, in one scenario, it is possible that the
duplex does not come into contact with RNase H, which implies (among
other possibilities) that this oligonucleotide may alter intracellular
mRNA transport. Alternatively, the duplex, perhaps because of steric
considerations, may not be recognized by RNase H.
Using a two-tailed, two-sample t test with unequal variances, no statistically significant difference was observed in MTT activity in PC-3 cells after treatments with 41 (co-down-regulation of Bcl-xL and Bcl-2) or 41m (down-regulation of Bcl-xL, no effect on Bcl-2) and either etoposide or carboplatin (P > 0.8). In addition, 41m in combination with paclitaxel was actually more inhibitory than 41 with paclitaxel (P = 10-6). In sharp contrast, however, 41m, in contrast to 41, did not sensitize PC-3 cells to mitoxantrone at all. These data imply that basal Bcl-2 expression, which in any case is much lower (at least 5–10-fold) in PC-3 cells than is Bcl-xL basal expression, is in general not highly protective to cytotoxic agents (mitoxantrone being an exception), and of the two antiapoptotic proteins, Bcl-xL is far more protective to a range of cytotoxics. This may not be the case, however, in cell lines engineered to overexpress Bcl-2 (5) or in other cell lines.
In summary, our data suggest that Bcl-xL is an extremely important protein in the set of pro- and antiapoptotic effector molecules that exist in prostate cancer cells, and the relative sensitivity of these cells to cytotoxic chemotherapy seems to be a direct function of the level of its expression. Our results also suggest that Bcl-2 may not be an optimum target in prostate cancer cells. In addition, experiments presented here further highlight the value of the antisense biotechnology, when used in what is hopefully an appropriate and rigorous manner, in the validation of gene function.
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FOOTNOTES |
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1 To whom requests for reprints should be
addressed, at Columbia University, 630 West 168 Street, New York, NY
10032. Phone: (212) 305-3606; Fax: (212) 305-7348; E-mail: stein{at}cuccfa.ccc.columbia.edu 
2 The abbreviations used are: FBS, fetal bovine
serum; mAb, monoclonal antibody; MTT,
(3-(4,5-dimethylthazol-2-yl)-2,5-diphenyl tetrazolium bromide; TAP,
tetra meso-(trimethylammonium)porphine; TMP, tetra
meso-(4-methylpyridyl)porphine; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase.
3 M. Vilenchik, N. Bander and C. A. Stein,
unpublished data.
Received 1/21/00. Accepted 9/ 1/00.
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