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Experimental Therapeutics |
Center for Surgery Research [S. M., Y. K., S. Kog., T. K., S. Kon.] and Department of Neurosurgery [T. K., S. Kon.], The Cleveland Clinic Foundation, Cleveland, Ohio 44195; Department of Neurosurgery, The Mount Sinai School of Medicine, New York, New York 10029-6574 [Y. K., T. K., S. Kon.]; and RammelKamp Center for Education and Research, MetroHealth Medical Center, Cleveland, Ohio 44109 [B. P. B.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, using an antisense oligonucleotide against hTER3 linked to 2-5A (5'-phosphorylated 2'-5'-linked oligoadenylate), we demonstrated its effect on cultured cells and s.c. tumors in nude mice of malignant glioma cell lines (8) . 2-5A is known as one pathway of IFN actions. 2-5A activates RNase L that is ubiquitous in mammalian cells, and this results in single-stranded RNA cleavage after UN (dinucleotide sequences beginning with a uridylate residue) sequences (9 , 10) . Antisense against a certain target linked to 2-5A causes the degradation of the targeted RNA in a highly specific manner and enhances the potency of antisense by at least 20-fold (11, 12, 13) . In the previous study, 2-5A-anti-hTER degraded hTER 5 h after the treatment and reduced glioma cell viability to 20–30% 2 weeks later (8) . The reduction of viability was mainly attributable to induction of apoptosis. Although the results were encouraging for the development of cancer therapy, our goal is to treat intracranial malignant gliomas with 2-5A-anti-hTER. Therefore, it is essential to investigate the effect of 2-5A-anti-hTER on intracranial tumors.
Recently, a liposome or cationic lipid has come into use to enhance the cellular incorporation of antisense oligonucleotides and is now very common for both in vitro and in vivo treatments (14, 15, 16, 17) . A cationic liposome is considered to bind negatively charged oligonucleotides or wrap them and enable them to enter into cells more easily and effectively (18) . In this study, Lipofectamine (Life Technologies, Inc., Grand Island, NY) was used to enhance the intracellular uptake efficacy of 2-5A-anti-hTER. As a result, it reduced both the concentration of 2-5A-anti-hTER and the frequency of treatment to yield much better effects than the results previously obtained without a cationic liposome. We here demonstrate that treatment with 2-5A-anti-hTER in the presence of Lipofectamine is significantly effective for intracranial malignant gliomas. The findings described in the present study suggest that 2-5A-anti-hTER is a promising therapeutic agent for the treatment of intracranial malignant gliomas expressing telomerase.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Synthesis of 2-5A-anti-hTER.
The selection of the antisense against hTER was described previously
(8)
. Briefly, we designed the 2-5A antisense
oligonucleotide to the region between residues 76 and 94, because this
part was predicted to be the most open by the MFOLD computer program.
The sequence of the antisense is 5'-GCG CGG GGA GCA AAA GCA C-3'. To
investigate the effect of 2-5A-anti-hTER, we synthesized a test
oligonucleotide (spA4-anti-hTER) with complete
homology to the targeted sequence and two controls (8)
.
One control is spA2-anti-hTER that has a
nonfunctional chimeric 2-5A linked to the anti-hTER. The other control
oligonucleotide is spA4-anti-(M6)hTER, which
contains functional 2-5A, but with six mismatched nucleotides in the
antisense that would prevent homologous binding with the telomerase
RNA. These chimeric oligonucleotides synthesized on solid supports and
purified as described previously (10, 11, 12, 13)
were gifts from
B. Bandyopadhyay and Dr. R. H. Silverman (Cleveland Clinic,
Cleveland, OH) or were synthesized at Hybridon (Milford, MA).
Cell Viability Assay.
The cytotoxic effect of 2-5A-anti-hTER on tumor cells was determined by
using a trypan blue dye exclusion assay as described previously
(8)
. The cells were seeded at 5 x 103
cells/well (0.1 ml) in 96-well flat-bottomed
plates and incubated overnight at 37°C. Then, oligonucleotides
(0.1–5.0 µM) with or without Lipofectamine (0.4–1.2
µl; Life Technologies, Inc.) were added into cells every 12 or
24 h. The first day of the treatment was counted as Day 0.
FITC-labeled 2-5A-anti-hTER.
FITC-labeled 2-5A-anti-hTER was a gift of Gemini Technologicals, Inc.
(Cleveland, OH). Cells (5 x 104)
were seeded in each well of 4-well chamber glass slides (Nalge Nunc
International, Naperville, IL). On the following day, the cells were
treated with 0.5 µM FITC-2-5A-anti-hTER with or without
Lipofectamine. After incubation for 3 h at 37°C, the cells were
washed with PBS at 4°C. Then, the cells were fixed with 4%
paraformaldehyde in PBS for 30 min on ice followed by washing with PBS
overnight. The cells were covered with Vectashield (Vector,
Burlingame, CA) and were coverslipped. The slides were observed and the
images were analyzed by the confocal laser scanning microscope
(Aristopan, Leica, Inc., Deerfield, IL) which belongs to the
core-facility of the Cleveland Clinic Foundation.
Telomerase Activity (TRAP) Assay.
The TRAP assay was performed with TRAPEZE Telomerase Detection kit
(Oncor Inc., Gaithersburg, MD) according to the manufacturer’s
instructions with some minor modifications (8
, 19)
. Cells
were washed once in PBS, and homogenized in 50 µl of ice-cold lysis
buffer. After 30 min of incubation on ice, the lysates were centrifuged
at 10,000 x g for 15 min at 4°C, and the
supernatant was rapidly frozen and stored at -80°C. The
concentration of protein was measured using the Bio-Rad Protein Assay
(Richmond, CA). Equal amounts of protein extracts were used for
telomerase assays. Two µl of each extract was assayed in a 50-µl
reaction mixture containing: 5 µl of 10x TRAP reaction buffer, 50
µM deoxynucleotide triphosphates, 0.2–0.4 µl
of [
-32P]dCTP (10 µCi/µl, 3000 Ci/mmol),
0.1 µg of TS primers, 1 µl of primer mix, 1 µg of T4 gene
32 protein (Boehringer Mannheim Biochemicals, Indianapolis, IN) and 2
units of Taq DNA polymerase (Boehringer Mannheim). An internal
telomerase activity standard (ITAS, 36 bp) was included in the primer
mix. After a 30-min incubation at room temperature for telomerase
extension, the reaction mixture was then subjected to PCR
amplification in a thermal cycler for 30 cycles at 94°C for
30 s, 50°C for 30 s, and 72°C for 1.5 min. The PCR
product was electrophoresed in 0.5 x Tris-borate
EDTA on a 6% polyacrylamide gel. The gel was then dried, and
autoradiography was performed. Radioactivity was also
quantitated with a Molecular Dynamics PhosphorImager
(Sunnyvale, CA).
Flow-Cytometric Analysis of Apoptosis.
When cells undergo apoptosis, one of the earliest changes is the
membrane phospholipid PS translocated from the inner to the
outer leaflet of the membrane. Thus, PS is exposed to the
external membrane and can be detected using PS-binding protein
such as annexin V (20)
. To provide a comparative assay of
apoptosis by annexin V labeling, tumor cells (1 x 106) treated with 2-5A-anti-hTER for 4 days were
harvested, washed, fixed with ice-cold 70% ethanol (50 min, 4°C) and
were resuspended in binding buffer [10 mM HEPES/NaOH (pH
7.4), 140 mM NaCl, and 2.5 mM
CaCl2]. Fifty µl of FITC-annexin V (R&D
Systems, Minneapolis, MN) were added and were incubated for 15 min in
the dark at room temperature before flow-cytometric analysis.
In Vivo Treatment.
For s.c. tumors, U373-MG cells (5.0 x 106 cells in 0.05 ml of serum-free DMEM and 0.05
ml of Matrigel) were inoculated s.c. into the right flank of
8–12-week-old female BALB/c nude mice (five mice for each treatment
group). Tumor growth was monitored by using calipers every 2 or 3 days.
Tumor volume (V) was calculated as (L x W2) ÷ 2, where
L = length (mm) and W = width (mm) as described previously (8)
.
Oligonucleotide treatment was initiated when the tumors reached a mean
tumor volume of 60–120 mm3
. We chose to treat
the animals after the establishment of the tumor to simulate the
clinical situation. spA4-anti-hTER or
spA4-anti-(M6)hTER (1–5 nmol/20 µl sterile
distilled water) with Lipofectamine (0–3.0 µl) were administrated by
injection directly into the tumor every 24 h or every other day
for 7 days. The percentage of increase in tumor volume was determined
compared with the initial tumor volume. To establish murine models with
intracranial tumors of U373-MG cells, BALB/c nude mice (8–12-week-old
female) were anesthetized using ketamine (90 mg/kg) and xylazine (10
mg/kg) and were placed in a stereotaxic frame (Lab Standard,
Stoelting, Wood Dale, IL) as described previously (21)
. A
burr hole was drilled in the skull 1.0 mm anterior and 2.0 mm lateral
to the bregma with a 0.9-mm burr to expose the dura. A microliter
syringe was fitted with a 27-gauge needle and connected to the
manipulating arm of the stereotaxic frame. Using it, we injected
5 x 105 tumor cells [10 µl of
a tumor cell suspension of 5 x 107/ml in serum-free DMEM (0.5 ml) and Matrigel
(0.5 ml)] transcranially at the depth of 4.0 mm from the drilled
hole. The needle was left in place for 1 min and then withdrawn
slowly over another minute. The scalp wound was closed with Autoclip
(Stoelting). Tumors that were infiltrating into surrounding normal
brain tissues were detected 2 weeks after the inoculation with tumor
cells (data not shown). To determine the effect of 2-5A-anti-hTER on
intracranial tumors, spA4-anti-hTER or
spA4-anti-(M6)hTER (3 nmol/10 µl distilled
water/1 µl Lipofectamine) was injected intracranially into the tumors
via the same stereotaxic approach 14, 16, 18, 21, and 23 days after the
inoculation of tumor cells. As controls, no-treatment and
Lipofectamine-alone (1 µl/10 µl distilled water) groups were
prepared. For histological analysis, mice were killed by cervical
dislocation, and the brains were removed and snap-frozen in liquid
nitrogen. The specimens were embedded in OCT compound, cut 7.0 µm in
thickness, and stained with H&E. For TUNEL assays, the frozen sections
were stained according to the manufacturer’s instructions (Oncor Inc.)
as described previously (8)
. During all of these
experiments, which were approved by the Cleveland Clinic
Foundation (CCF) Research Programs Committee, the animals were
housed and handled in accordance with NIH guidelines.
Statistical Analysis.
The data were expressed as means ± SD. Statistical
analysis was performed by using Student’s t test
(two-tailed). The significance of survival time differences between
treatment groups was evaluated using the Cox-Mantel test. The criterion
for statistical significance was taken as P < 0.05.
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RESULTS |
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First, we optimized the concentration of Lipofectamine to determine its
appropriate dose that, by itself, would not be cytotoxic to cells. As
shown in Fig. 1A
, we added Lipofectamine (0–1.2 µl/100 µl DMEM) to
U373-MG cells every 24 h for 4 days, and the cell viability of
tumor cells was determined. The cell viability of tumor cells was
decreased by the addition of Lipofectamine in a dose-dependent manner.
An amount of 0.8 or 1.2 µl of Lipofectamine reduced cell viability to
50 or 30%, respectively. However, the cytotoxic effect of 0.4 µl of
Lipofectamine was minimal. This, then, was the concentration chosen for
additional experiments.
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,
treatment with 2-5A-anti-hTER (2.0 or 5.0 µM)
without Lipofectamine every 12 h reduced cell viability to 75 or
45%, respectively. However, when the concentration of 2-5A-anti-hTER
was 0.1 or 0.5 µM, there was no antitumor
effect 4 days after the treatment of every 12 h. When
2-5A-anti-hTER (0.1–5.0 µM) was added every
24 h in the absence of Lipofectamine, the cytotoxic effect was not
observed. When Lipofectamine was mixed with 2-5A-anti-hTER (2.0 or 5.0
µM) and the cells were treated every 24 h,
the percentage of cell viability was 45 or 40%, respectively.
Interestingly, the lack of linearity of response of tumor cells to
increasing amounts of 2-5A-anti-hTER was observed, and the best effect
on cell viability was obtained when 0.5 µM
2-5A-anti-hTER mixed with Lipofectamine was added every 24 h. This
finding strongly suggested that to the extent that a cationic liposome
is used, the careful optimization of transfection conditions is
essential for the highest effect in 2-5A-anti-hTER as well as in
conventional DNA or antisense oligonucleotide.
The idea that a greater or lesser amount of 2-5A-anti-hTER or
Lipofectamine than the optimized ratio would impair its effect was also
supported by the findings obtained from s.c. tumor models as described
below. These results indicated that treatment with
2-5A-anti-hTER in the presence of Lipofectamine reduced the treatment
frequency to every 24 h and induced a better antitumor effect at
one-tenth of the concentration of oligonucleotides used previously. To
confirm that Lipofectamine enhanced the incorporation of 2-5A-anti-hTER
into tumor cells, U373-MG cells were treated with FITC-lableled
2-5A-anti-hTER. As shown in Fig. 1C
, tumor cells treated
with 2-5A-anti-hTER (0.5 µM) in the presence of
Lipofectamine for 3 h were all fluorescent-positive. In contrast,
any fluorescent-positive cells were undetectable when treated without
Lipofectamine. These results clearly indicated that Lipofectamine
enhanced the incorporation of 2-5A-anti-hTER into tumor cells.
Effect of 2-5A-anti-hTER on Malignant Glioma Cells in
Vitro.
We further investigated whether 2-5A-anti-hTER mixed with Lipofectamine
was effective for other malignant glioma cells. Prior to the cell
viability assay, we performed the TRAP assay to ensure that all of the
malignant glioma cells that we used were telomerase-positive and that
cultured astrocytes were telomerase-negative. As shown in Fig. 2A
, all of the malignant glioma cells (U373-MG,
U251-MG, GB-1, T98G, and
A172) expressed telomerase activity. In contrast, cultured
astrocytes P1N were telomerase-negative as described previously
(8)
. The internal telomerase activity standard
(ITAS) excluded the possibility of a false-negative result
attributable to Taq polymerase inhibitors.
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, spA4-anti-hTER
treatment with Lipofectamine every 24 h for 4 days reduced the
cell viability of tumor cells to 20–43%. In contrast, the cytotoxic
effect of control oligonucleotides
(spA2-anti-hTER and
spA4-anti-(M6)hTER) was not significant. On the
other hand, P1N astrocytes were resistant to those three types of
oligonucleotides even in the presence of Lipofectamine. Furthermore,
the flow-cytometric analysis using annexin V labeling was performed to
determine the extent of apoptosis quantitatively. As shown in Fig. 2C
, after the treatment with 2-5A-anti-hTER for 4 days, the
percentage of annexin V-binding apoptotic U373-MG cells was 3.88%
(a, Lipofectamine alone), 11.80% (b, control
oligonucleotide, spA4-anti-(M6)hTER, and
Lipofectamine), 14.92% (c, test oligonucleotide,
spA4-anti-hTER alone), or 99.82% (d,
spA4-anti-hTER and Lipofectamine), respectively.
The discrepancy of results between cell viability assays (20% of tumor
cells were viable) and apoptosis detection assays (99.82% of tumor
cells showed apoptosis) may derive from the possibility that some of
treated cells still remained viable while exhibiting apoptotic
features. These results indicated that the cytotoxic effect of
2-5A-anti-hTER in the presence of Lipofectamine was specifically for
telomerase-positive cells through induction of apoptosis, which
suggests that 2-5A-anti-hTER could work as a tool of targeted therapy
for telomerase-positive tumors.
Effect of 2-5A-anti-hTER on s.c. Tumors.
Obviously the goal in this study was to treat intracranial malignant
gliomas in mice. Before the experiments, we optimized the ratio of
2-5A-anti-hTER to Lipofectamine using s.c. tumors. At first, we treated
s.c. tumors with 5 nmol of 2-5A-anti-hTER, which was the same dose that
we injected previously (8)
. As shown in Fig. 3A
, treatment with 2-5A-anti-hTER (5 nmol) in the absence of
Lipofectamine reduced the tumor volume to 80% after 1 week. Next, s.c.
tumors were treated with 1 nmol of 2-5A-anti-hTER in the presence of
Lipofectamine (0 to 3 µl/each). As shown in Fig. 3A
, the
tumor volume was decreased to 47% by the treatment with 1 nmol of
spA4-anti-hTER and 0.3 µl of Lipofectamine
(P < 0.01, compared with treatment with 5
nmol of spA4-anti-hTER alone). The concentration
of 1 nmol of spA4-anti-hTER might be estimated to
correspond to about 20–30 times that used in cultured cells if
spA4-anti-hTER injected into the tumor should
cover the entire tumor. Although the situation between in
vitro and in vivo experiments is often different,
it might be necessary to investigate the in vivo antitumor
effect of the same concentration of 2-5A-anti-hTER as used in cultured
cells. Interestingly, the treatment at the ratio of
spA4-anti-hTER:Lipofectamine (1:0 or 1:3) showed
some inhibitory effects on tumor growth but did not regress the tumor
size.
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, there was no significant difference in treatment effect
between control oligonucleotide,
spA4-anti-(M6)hTER, and Lipofectamine alone. Less
frequent treatments with 2-5A-anti-hTER would be desirable, especially
for treating intracranial tumors, because direct treatments are
invasive. To investigate this possibility,
spA4-anti-hTER (3 nmol) mixed with Lipofectamine
(1 µl) was injected directly into the s.c. tumors every other day (a
total of four times) over a period of 7 days. As expected, this less
frequent treatment with higher concentration reduced the tumor volume
to 50% in size (data not shown). There was no significant difference
between spA4-anti-hTER at 1 nmol for seven
injections and at 3 nmol for four injections. These results indicated
that less frequent treatments with a higher concentration of
2-5A-anti-hTER might be feasible as the treatment modality of
intracranial tumors.
Effect of 2-5A-anti-hTER on Intracranial Tumors.
On the basis of the results of the s.c. tumors treatment, we decided to
treat intracranial tumors in mice with 3 nmol of 2-5A-anti-hTER and 1
µl of Lipofectamine by intracranial injections every other day (a
total of five times). Because intracranial tumors were clearly detected
2 weeks after the inoculation with malignant glioma cells (data not
shown), we started the treatment on day 14. We chose to treat the
animals after the establishment of the tumor to simulate the clinical
situation. As shown in Fig. 4A
, all of the control mice (the mice that received no
treatment or received Lipofectamine alone) with intracranial tumors
died of their tumors within 8–11 weeks. Intracranial tumors treated
with Lipofectamine alone grew extensively and invaded the other side of
the brain (Fig. 4, a and b
). Although
one of five mice treated with spA4-anti-(M6)hTER
survived up to 14 weeks after intracranial inoculation with tumor
cells, there was no statistical difference between control groups and
spA4-anti-(M6)hTER treatment. As shown in Fig. 4A
, one of five mice treated with
spA4-anti-hTER died of intracranial tumors 11
weeks after tumor inoculation, but four of five mice survived up to 15
weeks (P < 0.01, compared with control
groups). In brain tissues harvested from mice 15 weeks after treatment
with spA4-anti-hTER, the place where tumor cells
were inoculated and oligonucleotides were injected was detected, but
tumor tissues were not detected (Fig. 4, c and d
). To investigate whether apoptotic cells were detected in
the corresponding region, the TUNEL assay was performed using the
consecutive specimens. However, TUNEL-positive cells were not
detected (data not shown), which indicated the possibility that
apoptotic cells induced by 2-5A-anti-hTER treatment might be removed by
phagocytic cells until 15 weeks. These results indicated that the
treatment of mice bearing intracranial malignant gliomas with
2-5A-anti-hTER was therapeutically effective.
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DISCUSSION |
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It is expected that the inhibition of telomerase is a promising therapy for cancers because telomerase is detected in the vast majority of tumor cells but not in most normal cells (3, 4, 5 , 22) . Recent investigations show that telomerase activity is inhibited by treatment with the antisense oligonucleotides, hammerhead ribozyme, or peptide nucleic acids against hTER (23, 24, 25) . Actually, the above reports showed some effects on telomerase inhibition but not on cell viability. To investigate whether telomerase inhibition is available for cancer treatment, it is important to assess its effect on tumor growth using in vitro and in vivo systems. As demonstrated in this study, we have succeeded in treating tumor models in vitro and in vivo with 2-5A-anti-hTER. More recently, telomerase is reported to be essential for the formation of human tumor cells (26) . Therefore, our approach is expected to be effective for the treatment of a wide range of tumors including malignant gliomas.
Recently, three components of the human telomerase have been identified: the RNA component (hTER; Ref. 27 ), the telomerase-associated protein (TEP1; Refs. 28 , 29 ), and the telomerase catalytic subunit (hTERT; Refs. 30 , 31 ). Although both hTER and hTERT are necessary for telomerase activity, the expression of hTERT is well associated with telomerase activity (32 , 33) . That is to say, to treat telomerase-positive tumors with inhibition of telomerase, it is likely that the hTERT is more attractive as a target than the hTER. Actually, the expression of the dominant-negative mutants of hTERT resulted in complete inhibition of telomerase activity, reduction in telomere length, and death of tumor cells (34 , 35) . This cell death was telomere-length dependent, inasmuch as cells with a long telomere were viable but exhibited telomere shortening at a rate similar to that of mortal cells (34 , 35) . The cell death in cells with a short telomere was induced 5–10 days after the induction of a dominant-negative hTERT mutant (35) . In contrast, the present treatment with 2-5A-anti-hTER induced massive apoptosis just 4 days after the treatment. What is the molecular mechanism by which treatment with 2-5A-anti-hTER induces apoptosis in tumors within only 4 days? To answer the question, the following two observations, which are still under study, may be useful: (a) it took approximately 1 month to induce apoptosis in U251-MG or HeLa cells by telomere shortening after telomerase inhibition (19 , 27) . When the telomere length reached the critical length, apoptosis was triggered. On the other hand, U251-MG cells in the present study and HeLa cells (in ongoing studies)4 underwent apoptosis in more than 50% of cells within 4 days after 2-5A-anti-hTER treatment. This raises the possibility that 2-5A-anti-hTER may directly or indirectly stimulate the apoptotic pathways regardless of telomere length in tumors; and (b) we have obtained evidence, using the caspase inhibition assays, that the caspase family is involved in the cytotoxic effect of 2-5A-anti-hTER on malignant glioma cells4 or on prostate cancer cells (36) . Caspases play a major role in the transduction of apoptotic signals and in the execution of apoptosis in mammalian cells (37, 38, 39, 40, 41) . Therefore, caspases are thought to be a key factor in the function of 2-5A-anti-hTER. Although additional studies are necessary to ascertain precisely the molecular mechanism underlying the effect of 2-5A-anti-hTER, we will be able to conclude that its effect is attributable to the activation of the apoptosis pathway involving the caspase family irrespective of telomere length.
In summary, the treatment with 2-5A-anti-hTER that was modified by the use of a cationic liposome effectively suppressed growth of cultured cells and s.c. tumors of malignant gliomas. Furthermore, treatment of intracranial malignant gliomas with 2-5A-anti-hTER was therapeutically effective. This is the first report demonstrating the feasibility of antisense telomerase therapy for intracranial tumors. The present study strongly indicates that 2-5A-anti-hTER is a promising experimental therapeutic agent for the treatment of malignant gliomas with telomerase activity.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This study was supported in part by Cleveland
Clinic Foundation Research Fund No. 5928 (to S. Kon.), the
John Gagliarducci Fund (to S. Kon.), and the USPHS Grant
1R01CA80233 (to S. Kon.) awarded by the National Cancer
Institute. 
2 To whom requests for reprints should be
addressed, at Department of Neurosurgery, The Mount Sinai Medical
Center, One Gustave L. Levy Place, Box 1136, New York, NY 10029-6574.
Phone: (212) 241-6503; Fax: (212) 831-3324; E-mail: Seiji.Kondo{at}mssm.edu
3 The abbreviations used are: hTER, human
telomerase RNA; anti-hTER, antisense hTER; TRAP, telomeric repeat
amplification protocol; PS, phosphatidylserine; TUNEL, terminal
deoxynucleotidyl transferase (Tdt)-mediated nick end labeling; hTERT,
human telomerase reverse transcriptase.
Received 11/22/99. Accepted 6/20/00.
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B signaling by selective ablation of an mRNA target by 2-5A antisense chimeras. Science (Washington DC), 265: 789-792, 1994.This article has been cited by other articles:
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 W. C. Hahn Role of Telomeres and Telomerase in the Pathogenesis of Human Cancer J. Clin. Oncol., May 15, 2003; 21(10): 2034 - 2043. [Abstract] [Full Text] [PDF]
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 J.-L. Mergny, J.-F. Riou, P. Mailliet, M.-P. Teulade-Fichou, and E. Gilson Natural and pharmacological regulation of telomerase Nucleic Acids Res., February 15, 2002; 30(4): 839 - 865. [Abstract] [Full Text] [PDF]
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R. Pruzan, K. Pongracz, K. Gietzen, G. Wallweber, and S. Gryaznov Allosteric inhibitors of telomerase: oligonucleotide N3'->P5' phosphoramidates Nucleic Acids Res., January 15, 2002; 30(2): 559 - 568. [Abstract] [Full Text] [PDF]
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