Telomerase antagonists GRN163 and GRN163L inhibit tumor growth and increase chemosensitivity of human hepatoma
Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation. Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma. GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat flank xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a dose-dependent manner in vitro and in vivo. The potency and efficacy of the lipid-conjugated antagonist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction, reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the development of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma, GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.
Telomerase Antagonists GRN163 and GRN163L Inhibit
Tumor Growth and Increase Chemosensitivity of
Meta W. Djojosubroto,
Allison C. Chin,
Michael P. Manns,
Calvin B. Harley,
and K. Lenhard Rudolph
Most cancer cells have an immortal growth capacity as a consequence of telomerase reactivation.
Inhibition of this enzyme leads to increased telomere dysfunction, which limits the proliferative
capacity of tumor cells; thus, telomerase inhibition represents a potentially safe and universal target for
cancer treatment. We evaluated the potential of two thio-phosphoramidate oligonucleotide inhibitors
of telomerase, GRN163 and GRN163L, as drug candidates for the treatment of human hepatoma.
GRN163 and GRN163L were tested in preclinical studies using systemic administration to treat ﬂank
xenografts of different human hepatoma cell lines (Hep3B and Huh7) in nude mice. The studies
showed that both GRN163 and GRN163L inhibited telomerase activity and tumor cell growth in a
dose-dependent manner in vitro and in vivo. The potency and efﬁcacy of the lipid-conjugated antag-
onist, GRN163L, was superior to the nonlipidated parent compound, GRN163. Impaired tumor
growth in vivo was associated with critical telomere shortening, induction of telomere dysfunction,
reduced rate of cell proliferation, and increased apoptosis in the treatment groups. In vitro, GRN163L
administration led to higher prevalence of chromosomal telomere-free ends and DNA damage foci in
both hepatoma cell lines. In addition, in vitro chemosensitivity assay showed that pretreatment with
GRN163L increased doxorubicin sensitivity of Hep3B. In conclusion, our data support the develop-
ment of GRN163L, a novel lipidated conjugate of the telomerase inhibitor GRN163, for systemic
treatment of human hepatoma. In addition to limiting the proliferative capacity of hepatoma,
GRN163L might also increase the sensitivity of this tumor type to conventional chemotherapy.
epatocellular carcinoma (HCC) is one of the
most prevalent cancers worldwide,
and the in-
cidence of HCC in developed countries is still
increasing as a result of a growing number of carriers of
chronic hepatitis C virus infection.
The therapeutic op-
tions for advanced-stage HCC are limited, and this cancer
responds poorly to systemic treatment with chemother-
apy. Other therapeutic strategies have not revealed signif-
icant efﬁciency in clinical trials (reviewed by Llovet et
). Therefore, there is currently no standard therapy for
advanced, multifocal HCC, indicating the need to evalu-
ate new therapeutic options for this tumor.
Similar to other malignant human tumor types,
80% of human HCC biopsies show activation of telom-
In contrast, most somatic human tissues, including
show no or very low levels of telomerase
activity. The main function of telomerase is the de novo
synthesis of telomeres, which cap the chromosome ends
of eukaryotic cells and protect chromosome ends from
fusion and DNA damage recognition.
Because of the
“end replication problem” of DNA polymerase, telomeres
shorten during each cell division by 50 to 100 bp.
mere shortening to a critical length and/or the uncapping
of the telomere limit the growth of primary human cells to
a ﬁnite number of cell divisions, leading to either replica-
tive senescence or crisis.
A critically short telomere
Abbreviations: HCC, hepatocellular carcinoma; NPS, N3⬘-P5⬘ thio-phosphor-
amidate; PBS, phosphate-buffered saline; bw, body weight; BrdU, 5-bromo-2-
deoxyuridine; TUNEL, terminal deoxynucleotidyl transferase–mediated dUTP
nick end labeling; Q-FISH, quantitative ﬂuorescence in situ hybridization; TFI,
telomere ﬂuorescence intensity; TRAP, telomere repeat ampliﬁcation protocol; TRF,
telomere restriction fragment.
Department of Gastroenterology, Hepatology, and Endocrinology,
Medical School Hannover, Hannover, Germany; and the
Menlo Park, CA.
Received December 6, 2004; accepted June 9, 2005.
K.L.R. is supported by the Deutsche Forschungsgemeinschaft (Emmy-Noether-
Programm: Ru 745/2-3 and KFO119 and Ru 745/4-1) and by a grant from the
Deutsche Krebshilfe e.V. (10-2236-Ru2).
Address reprint requests to: K. Lenhard Rudolph, Department of Gastroenterol-
ogy, Hepatology, and Endocrinology, Medical School Hannover, Carl-Neuberg-Str.
1, 30625 Hannover, Germany. E-mail: email@example.com; fax:
Copyright © 2005 by the American Association for the Study of Liver Diseases.
Published online in Wiley InterScience (www.interscience.wiley.com).
Potential conﬂict of interest: Drs. Chin, Go, Gryaznov, and Harley own stock in
within a cell is believed to activate DNA damage signal-
ing, inducing senescence or cell death.
It has been
proposed that postnatal repression of telomerase func-
tions as a tumor repressor mechanism that limits the
growth of transformed cells.
This hypothesis is consis-
tent with the high frequency of telomerase reactivation
found in most human cancers, including HCC. Studies
on the inhibition of telomerase activity in cancer cell
and in telomerase knockout mice
shown that growth and progression of malignant tumors
depend on telomerase activity and telomere stabilization.
Recently, a new class of telomerase inhibitors was de-
veloped: N3⬘-P5⬘ thio-phosphoramidate (NPS) oligonu-
cleotides targeting the active site template region of the
human telomerase RNA component.
revealed that a development candidate from this class of
oligonucleotides, GRN163, inhibited telomerase activity
in various cultured human cancer cell lines (breast, renal,
prostate, epidermoid, cervix, lung, colon, leukemia, mul-
tiple myeloma, lymphoma) leading to telomere shorten-
ing, growth arrest, and apoptosis.
GRN163 inhibited tumor growth of prostate cancer,
multiple myeloma, lymphoma, and glioblastoma in xeno-
To evaluate the potential use of
NPSS oligonucleotides targeting the human telomerase
RNA component for the treatment of HCC, we analyzed
GRN163 and GRN163L, a lipid-conjugated derivative of
GRN163 designed for increased bioavailability,
titumor effects against human hepatoma cells (Hep3B
and Huh7) in vivo and in vitro. The results suggest that
telomerase inhibition could be a valid approach for HCC
treatment and that GRN163L is a promising drug devel-
opment candidate with signiﬁcant effects on tumor
growth and increased chemosensitivity to doxorubicin.
Materials and Methods
Mouse Handling. NMRI nu/nu mice were bred and
housed at the animal facility of Medical School Han-
nover, given a standard diet, and placed on a 12/12-hour
light/dark cycle in a pathogen-free barrier area. Protocols
used in this study complied with institutional guidelines.
Mice were humanely sacriﬁced when the total two-di-
mensional size of tumors exceeded 40 mm or after the
treatment period of 4 weeks.
Culture and Subcutaneous Inoculation of Cells.
The human hepatoma cell lines Hep3B and Huh7 were
purchased from American Type Culture Collection. Cells
were cultured in Dulbecco’s modiﬁed Eagle medium
(Gibco, Grand Island, NY) supplemented with 10% fetal
bovine serum (Gibco) and penicillin/streptomycin (100
g/mL; Biochrom, Cambridge, UK ) at 37°C and
. Cells were passaged every 2 days (Huh7) and 3
to 4 days (Hep3B) 2 to 3 times before being inoculated
into nude mice. Mice were anesthetized via methyl-ether
inhalation, and 5 ⫻ 10
hepatoma cells were engrafted
subcutaneously into the right and left ﬂanks.
Test Articles. GRN163 is a 13-mer thio-phosphor-
amidate oligonucleotide with the sequence 5⬘-
TAGGGTTAGACAA. GRN163L is a lipid-conjugated
derivative of GRN163 with the sequence 5⬘-L-
TAGGGTTAGACAA, where L ⫽ aminoglycerol-palmi-
toyl moiety. Lyophilized powders for GRN163 and
GRN163L were formulated in phosphate-buffered saline
(PBS) at 5.0 and 3.3 mg/mL, respectively, using an ex-
tinction coefﬁcient of 143 OD/
mol. Formulated oligo-
nucleotide compounds were ﬁlter-sterilized (0.2
and stored in 5- to 10-mL aliquots at ⫺20°C.
Treatment Groups and Dosing Regimens. Treat-
ment via intraperitoneal injection of vehicle or drug was
started when a subcutaneous tumor became detectable
(day 1). Mice were randomized into groups (n ⫽ 5-7
mice/group for Hep3B groups; n ⫽ 13-18 mice/group for
Huh7 groups). In the ﬁrst round of treatment of Hep3B-
derived tumors, we injected the control group of mice
with PBS and three treatment groups with 30 mg/kg body
weight (bw) GRN163, 30 mg/kg bw GRN163L, and 10
mg/kg bw GRN163L (n ⫽ 5-7 mice/group); dose admin-
istration was 5 times a week (Monday to Friday). In the
second round of study on Huh7-derived tumors, mice
were assigned to two groups—control (PBS) and
GRN163L (30 mg/kg bw)—and injections were given 5
times a week (Monday to Friday). In the third round of
treatment of Hep3B-derived tumors, mice were assigned
to ﬁve groups— control (PBS), GRN163 (50 mg/kg bw),
GRN163 (16.7 mg/kg bw), GRN163L (18.3 mg/kg bw),
and GRN163L (6.1 mg/kg bw)—and the injections were
given 3 times a week (Monday, Wednesday, and Friday).
Tumor length and width were measured daily; tumor size
was calculated using the formula v ⫽ (lw
)/2. Mice in
which the total two-dimensional size of tumors had not
grown beyond 40 mm were humanely sacriﬁced via CO
inhalation after completion of the treatment period of 4
weeks, typically 18 to 24 hours after the last dosing. Tu-
mor samples were collected and snap-frozen in liquid ni-
Cell Proliferation Assay. Cell proliferation assay via
5-bromo-2-deoxyuridine (BrdU) incorporation was per-
formed on frozen sections of tumors as described else-
The BrdU-labeling index was determined by
counting the number of BrdU-positive cells under low-
power magniﬁcation (100⫻) ﬁelds in a blinded manner.
Total positive cells from at least 55 ﬁelds/group were
1128 DJOJOSUBROTO ET AL. HEPATOLOGY, November 2005
counted in Hep3B tumors treated with PBS and with 30
mg/kg bw GRN163L.
In Situ Cell Death Detection. Apoptosis analysis was
performed via terminal deoxynucleotidyl transferase–me-
diated dUTP nick end labeling (TUNEL) assay. Analysis
was performed on frozen sections of tumor samples ac-
cording to a protocol provided in the In Situ Cell Death
Detection Kit (Roche, Mannheim, Germany). Slides
were mounted with Vectashield mounting solution (Vec-
tor Laboratories, Gru¨nberg, Germany) containing DAPI.
Apoptotic areas of the tumors were classiﬁed as ⬍10%,
10%-20%, or ⬎20% according to the density of
TUNEL-positive cells under low-power magniﬁcation
(100⫻) ﬁelds. In total, at least 50 ﬁelds/group were scored
in tumors treated with PBS and with 30 mg/kg bw
GRN163L. All counts were performed in a blinded man-
Anaphase Bridge Index. Hep3B and Huh7 tumors
were ﬁxed in 4% formaldehyde, embedded in parafﬁn,
and subjected to hematoxylin-eosin staining. The an-
aphase bridge index was calculated by dividing the num-
ber of anaphase bridges by the total number of anaphases
in 4 to 5 tumors from four groups (Hep3B, PBS control
and GRN163L-treated [30 mg/kg bw]; Huh7, PBS con-
trol and GRN163L-treated [30 mg/kg bw]).
Telomere-Speciﬁc Quantitative Fluorescence In
Situ Hybridization. Quantitative ﬂuorescence in situ
hybridization (Q-FISH) for telomere ends was performed
as described elsewhere.
Telomere ﬂuorescence intensity
(TFI) was quantiﬁed by using TFL-TELO version 1.0a
We observed TFI ranging from 100 to 2,800,
and these values were binned into groups of 1-100, 101-
200, and so forth. In total, ﬂuorescence intensities of an
average of 50 cells/tumor from ﬁve control tumors and six
tumors treated with 30 mg/kg bw GRN163L were quan-
Telomerase Extract Preparation and Telomere Re-
peat Ampliﬁcation Protocol Assay. Telomerase extrac-
tion and telomere repeat ampliﬁcation protocol (TRAP)
assays were performed with the TRAPeze Telomerase De-
tection System (Chemicon, Hampshire, UK) according
to the manufacturer’s instructions. Brieﬂy, a small por-
tion of the tumor was minced in ice-cold PBS, incubated
in CHAPS lysis buffer containing 200 U/mL RNAse in-
hibitor (Sigma, Munich, Germany) on ice, and centri-
fuged at 12,000g. Two hundred nanograms of
supernatant containing telomerase extract was used per
reaction for the TRAP assay. The tumor cell extract pro-
vided by the manufacturer was used as a positive control,
and the heat-inactivated extract of the same batch was
used as a negative control. TRAP mixtures were subjected
to telomerase extension at 30°C for 30 minutes and then
to polymerase chain reaction ampliﬁcation in the pres-
ence of telomerase substrate (TS) primer labeled with
ATP (Amersham Biosciences, Freiburg, Germany).
TRAP products were size-fractioned on 16% polyacryl-
amide gels. TRAP product ladders were quantiﬁed using a
custom image quantitation program that extracts the
chromatogram proﬁle from each lane and sums the area
under the peaks.
Metaphase Spreads Preparation and Staining.
Metaphase spreads were prepared from cells treated with
mol/L GRN163L for 21 days (Hep3B) and 35 days
(Huh7). Cell cultivation was performed as described
above. Demecolsine (0.2
g/mL [Sigma]) was adminis-
tered 10 hours before collection of cells. Cells were incu-
bated in 0.075 mol/L KCl for 40 minutes at 37°C, ﬁxed,
and washed in methanol/acetic acid (3:1) several times
before being trickled onto slides. Telomeres were visual-
ized using telomere-speciﬁc Q-FISH as described in Te-
lomere-Speciﬁc Quantitative Fluorescence In Situ
Hybridization and chromosomes were counterstained
with DAPI. Telomere-free ends were scored in at least 30
metaphases per sample.
DNA Damage Protein Staining.
53bp1 staining was performed on cultivated Hep3B and
Huh7 cells treated with 10
mol/L GRN163L for 40 and
70 days, respectively. Cells were grown on sterile cover
slips overnight at subconﬂuent density. Before antibody
staining, cells were washed with PBS, ﬁxed in 1% form-
aldehyde, and blocked in normal goat serum. Primary
-H2ax (1:250 [Bethyl Labs, Montgomery,
TX]) or anti-53bp1 (1:250 [Abcam, Cambridge, UK])
and secondary Cy3-conjugated goat anti-rabbit immuno-
globulin G (1:1000 [Sigma]) were used to probe and vi-
-H2ax and 53bp1 foci, respectively. DNA
damage foci were counted in a total of 300 nuclei per
Telomere Restriction Fragment Length Analysis.
Telomere restriction fragments (TRFs) were determined
in DNA isolated from cells cultivated for 35 days
(Hep3B) and 70 days (Huh7), with and without the pres-
ence of 10
mol/L GRN163L. TRF length analysis was
performed as previously described.
In Vitro Combination Treatment With Doxorubi-
cin. Hep3B cells were cultured in Eagle minimal essen-
tial medium containing 10% fetal bovine serum, 0.1
mmol/L nonessential amino acids, and 0.1 mmol/L so-
dium pyruvate (all from Gibco). The cells were passaged 3
times a week. At each passage, 6 to 8 ⫻ 10
seeded into 75-cm
ﬂasks. Hep3B cells in log-growth
phase were pretreated with GRN163L at 0.1, 1, 3, and 10
mol/L for 5 to 14 days in cell culture ﬂasks. Fresh
GRN163L-containing medium was replenished at each
HEPATOLOGY, Vol. 42, No. 5, 2005 DJOJOSUBROTO ET AL. 1129
passage. One day before doxorubicin treatment, Hep3B
cells were reseeded in GRN163L-containing medium at a
subconﬂuent density (6,000 cells/well in a 96-well dish).
Doxorubicin was added to the medium at ﬁnal concen-
trations of 10 to 10,000 nmol/L, and cells were incubated
for an additional 24 to 48 hours. Cell viability was mea-
sured via XTT assay using the Cell Proliferation Kit II
Statistical Analysis. Statistical analysis was accom-
plished using Microsoft Excel and GraphPad Prism 3.0
(GraphPad Software, Inc.). A two-tailed Student t test
with unequal variance and ANOVA were used to calcu-
late the P values of tumor volumes. Telomeric Q-FISH
median was calculated via cumulative addition, and the P
value was calculated using a
test. P values for other
assays were calculated using a Student t test. In all assays,
P values of less than .05 and .001 were considered statis-
tically signiﬁcant and highly signiﬁcant, respectively.
GRN163 and GRN163L Inhibit Tumor Growth of
Human Hepatoma After Xenotransplantation. Hepa-
toma appeared between days 14 and 30 after subcutane-
ous injection of 5 ⫻ 10
Hep3B cells in approximately
70% of the injected nu/nu mice, and between days 7 and
10 after injection of 5 ⫻ 10
Huh7 cells in approximately
90% of the injected nu/nu mice (data not shown). After
subcutaneous hepatoma became visible, the mice were
grouped into cohorts, and treatment with the indicated
doses of GRN163, GRN163L, or PBS control (Fig. 1)
was started (day 1). In the ﬁrst and second experiments
with Hep3B- and Huh7-derived tumors, mice were
treated 5 times a week (Monday to Friday) (Fig. 1A-B).
During the third experiment with Hep3B-derived tu-
mors, mice were treated 3 times a week (Monday,
Wednesday, and Friday) and at lower concentrations to
deﬁne the minimum effective dose. In the ﬁrst experi-
ment, both NPS-oligonucleotides (GRN163 and
GRN163L) showed antitumor activity resulting in signif-
icant reduction in tumor growth in the treatment groups
compared with the control group at days 18 and 27 (Fig.
1A). The late time points showed a dose-dependent re-
sponse for GRN163L, with signiﬁcantly stronger inhibi-
tion of tumor growth for mice treated with 30 mg/kg bw
per injection compared with mice receiving 10 mg/kg bw
per injection (Fig. 1A). Inhibition of tumor growth was
observed earlier in the GRN163L-treated groups with
Huh7-derived tumors compared with control mice (Fig.
1B). In the third experiment, we observed a delayed anti-
tumor activity at day 27 of treatment, and only
GRN163L (18.3 mg/kg bw) induced a signiﬁcant reduc-
tion of tumor growth (Fig. 1C). In contrast, GRN163 (at
50 mg/kg bw and 16.6 mg/kg bw) as well as GRN163L
(at 6.1 mg/kg bw) did not show signiﬁcant inhibition of
tumor growth. Thus, in this model it appears that weekly
doses of 150 mg/kg of GRN163 or 50 mg/kg of
GRN163L are sufﬁcient for efﬁcacy, and that a dosing
regimen of three applications per week is less effective
than ﬁve applications per week. Further work will be re-
quired to fully deﬁne the impact of dose and schedule on
tumor growth suppression. The overall smaller tumor size
in all groups at day 27 of the third experiment compared
with that of the ﬁrst experiment correlated with reduced
starting tumor sizes in the cohorts of the third experiment
compared with the ﬁrst experiment. No obvious toxicity,
weight loss, or other signs of morbidity were observed in
any treatment group.
GRN163L Inhibits Telomerase Activity in Hepa-
toma Cells In Vitro and in Transplanted Hepatoma
In Vivo. To understand the mechanism of tumor growth
inhibition by GRN163L, we ﬁrst monitored telomerase
Fig. 1. Administration of GRN163 and GRN163L restrained the growth
of tumors in a dose-dependent manner. Histograms show average tumor
volume (v ⫽ lw
/2 ⫾ SEM, in mm
) for control and treatment groups
from the (A) ﬁrst and (C) third round in 9-day intervals (n ⫽ 5-7
mice/group) and the (B) second round in 3-day intervals (n ⫽ 13-18
mice/group). Treatment was administered 5 times a week (Monday to
Friday) during the ﬁrst and second rounds and 3 times a week (Monday,
Wednesday, and Friday) during the third round. Note that in the second
round, only GRN163L showed signiﬁcant effect of treatment, which might
relate to the different dosing regimen. PBS, phosphate-buffered saline.
1130 DJOJOSUBROTO ET AL. HEPATOLOGY, November 2005
activity in the Hep3B and Huh7 cell lines that were used
for our xenotransplant experiments, as well as in another
hepatoma cell line, HepG2. This analysis showed that
GRN163L inhibited telomerase activity in Hep3B,
Huh7, and HepG2 cells with an IC
mol/L, and 0.63
mol/L, respectively (Fig. 2A-C).
We next analyzed telomerase activity in xenotransplanted
Hep3B- and Huh7-derived hepatoma treated with
GRN163L or PBS as a control. TRAP assay revealed 42%
(P ⬍ .001) and 34% (P ⬍ .05) reductions of telomerase
activity in GRN163L-treated Hep3B and Huh7 tumors,
respectively, as compared with those in tumors from con-
trol mice (Fig. 3A).
Because antitumor activity of telomerase inhibition has
previously been linked to telomere length,
telomere length in GRN163L-treated and PBS-treated
control Hep3B tumors. Telomere length measurements
were performed using Q-FISH, which analyzes ﬂuores-
cent signals of telomeres after hybridization with a te-
lomere-speciﬁc probe giving an intensity that corresponds
to telomere length.
Our analysis revealed a signiﬁcant
increase in nuclei with very low total ﬂuorescence inten-
sity (TFI ⬍ 400) in Hep3B tumors treated with
GRN163L compared with those from control mice
treated with PBS (Fig. 3B). The wide range of TFI in
these xenotransplanted tumors, especially the occurrence
of very strong TFIs, could be due to inﬁltration of host
cells (epithelial cells, ﬁbroblasts) into the transplanted
hepatoma cells. Histological analysis via hematoxylin-eo-
sin staining did not reveal any signiﬁcant difference in the
composition of transplanted hepatoma between the dif-
ferent groups. All tumors consisted of approximately 80%
hepatoma cells (data not shown).
TRF Length Analysis. To determine whether telo-
mere length of treated Hep3B and Huh7 cells might be
maintained by an alternative lengthening of telomeres
mechanism, TRF lengths of Hep3B and Huh7 cells were
analyzed in DNA isolated from cells cultivated with and
without addition of 10
mol/L GRN163L for extended
periods. After administration of 10
for 35 and 70 days for Hep3B and Huh7, respectively, the
average TRF length of both cell lines decreased, and there
was no evidence of increased heterogeneity in length char-
acteristic of alternative lengthening of telomeres (Fig.
3C). Moreover, growth rates were suppressed (data not
shown), and there was no indication that cells escaped
from antitelomerase treatment. Together, these data sug-
Fig. 3. (A) Telomerase activity showed an approximately 42% reduc-
tion in Hep3B tumors and and a 34% reduction in Huh7 tumors treated
with 30 mg/kg bw GRN163L compared with controls (n ⫽ 4 tumors/
group). (B) GRN163L-treated Hep3B tumors showed more cells with
critically short telomeres (10.9% vs. 5.2% in those of the control group;
P ⬍ .05) via Q-FISH analysis. Dashed lines show the median telomere
length. Analysis was performed using telomere-speciﬁc Q-FISH. (C) TRF
length analysis using in-gel Southern hybridization shows shorter telo-
meres in cultivated Hep3B and Huh7 cells treated with 10
GRN163L for 35 and 70 days, respectively. Dashed lines show the mean
Fig. 2. Signiﬁcant reduction of telomerase activity by GRN163L cor-
relating with shortened telomeres. GRN163L inhibited telomerase activity
in (A) Hep3B, (B) Huh7, and (C) HepG2 cells with an IC
mol/L, and 0.63
mol/L, respectively. IC
calculated automatically by ﬁtting the data points to a dose–response
curve using nonlinear regression with constant values for the top (100%)
and bottom (0%) (Prism).
HEPATOLOGY, Vol. 42, No. 5, 2005 DJOJOSUBROTO ET AL. 1131
gest that alternative lengthening of telomeres was not en-
gaged in the maintenance of telomere length in these cell
lines in response to telomerase inhibition. Interestingly,
the mean telomere length of Huh7 cell was longer com-
pared with Hep3B cells, yet the xenograft tumors of
Huh7 cells showed an early response to telomerase inhi-
bition (Fig. 1B)—indicating that, in addition to telomere
length, other factors had an impact on sensitivity of tumor
cells in response to telomerase inhibition.
GRN163L Inhibits Tumor Cell Proliferation, In-
creases Tumor Cell Apoptosis, and Leads to Forma-
tion of Anaphase Bridges. To evaluate the correlation
between shortened telomeres and tumor cell prolifera-
tion rates, we performed BrdU incorporation assay on
tumors treated with 30 mg/kg bw GRN163L and with
PBS. Hep3B tumor sections contained 36.5% less
BrdU-positive cells after treatment with 30 mg/kg bw
GRN163L compared with the PBS-treated controls
(145.0 ⫾ 33.7 vs. 92.0 ⫾ 40.0 positive cells/low power
ﬁeld; P ⬍ .001) (Fig. 4A). In addition, TUNEL assays
revealed an increased incidence of apoptosis in
GRN163L-treated tumors (Fig. 4B), which is in accor-
dance with previous in vitro studies with GRN163.
Using hematoxylin-eosin staining, we observed a
higher percentage of anaphase bridges—a hallmark of
telomere dysfunction—in the Hep3B and Huh7 tu-
mors treated with 30 mg/kg bw GRN163L compared
with PBS-treated controls (Fig. 4C).
Increase of Telomere-Free Ends and DNA Damage
Signals in Hepatoma Cells Treated With GRN163L.
The ﬁnding on anaphase bridge formation indicated
that telomerase inhibition by GRN163L resulted in
telomere dysfunction in hepatoma cells. To directly
test this hypothesis, we analyzed metaphase prepara-
tions from Hep3B and Huh7 cells cultivated in vitro
with and without the presence of 10
GRN163L. The treated cells showed a signiﬁcantly
higher number of chromosomes with telomere-free
ends compared with the untreated cells (Fig. 5A). The
presence of more telomere-free ends in cells treated
with GRN163L correlated with an increased frequency
-H2AX and 53bp1 foci (Fig. 5B-C), two molecular
markers previously demonstrated to localize to sites of
DNA strand breaks and dysfunctional telomeres.
GRN163L Increases In Vitro Chemosensitivity of
Hep3B Cells to Doxorubicin. To explore the possibil-
Fig. 4. Lower proliferation rate,
increased tendency toward apopto-
sis, and more anaphase bridge for-
mation in tumors treated with
GRN163L. (A) The left panel shows
the average number of BrdU-positive
cells in the control and treatment
groups. The right panel shows rep-
resentative photographs of the ﬁelds
counted (original magniﬁcation,
100⫻). (B) The left panel shows the
percentage of ﬁelds with the speci-
ﬁed percent TUNEL-positive cells in
control and treated groups. The right
panel shows representative photo-
graphs of the ﬁelds scored (original
magniﬁcation, 100⫻). (C) Histo-
grams in the left panel show the
percentage of anaphase-bridges ob-
served in 50 mitotic cells/sample
(n ⫽ 4-5 tumors/group). The right
panel shows representative pho-
tomicrographs from anaphase
bridges observed in Hep3B and
Huh7 tumors. BrdU, 5-bromo-2-de-
oxyuridine; PBS, phosphate-buffered
saline; TUNEL, terminal deoxynu-
cleotidyl transferase–mediated dUTP
nick end labeling.
1132 DJOJOSUBROTO ET AL. HEPATOLOGY, November 2005
ity of enhancing the antitumor effect of GRN163L, we
tested an in vitro combination treatment of GRN163L
and the chemotoxic agent doxorubicin. In this study,
Hep3B cells were pretreated with 0.1, 1, 3, and 10
mol/L of GRN163L for 5 to 14 days before doxoru-
bicin was added to the medium at a ﬁnal concentration
of 10 to 10,000 nmol/L and incubated for 24 to 48
hours. The XTT cell viability assay showed that pre-
treatment with 1
mol/L GRN163L increased the che-
mosensitivity of Hep3B cells (Fig. 6) and signiﬁcantly
Fig. 5. Induction of telomere dysfunction in hepatoma cells treated with GRN163L. Treatment of in vitro cultured hepatoma cells with 10
GRN163L for the indicated periods led to (A) a signiﬁcantly higher prevalence of telomere-free ends in metaphase spreads of Hep3B and Huh7 cells
and an increased number of DNA damage
-H2ax and 53bp1 foci in (B) Huh7 and (C) Hep3B cells. The photomicrographs to the right of the
histograms show representative images of (A) chromosomes and (B-C) DNA damage foci.
HEPATOLOGY, Vol. 42, No. 5, 2005 DJOJOSUBROTO ET AL. 1133
lowered the LD
of doxorubicin in Hep3B cells (Table
1). Cells pretreated with 0.1
mol/L GRN163L be-
haved similarly to untreated cells, while cells pretreated
with higher concentration of GRN163L (3 and 10
mol/L) did not replate efﬁciently (data not shown).
These data strongly suggest an enhanced antitumor
effect via a combination treatment of GRN163L and
Our study provides experimental evidence that telom-
erase inhibition by NPS oligonucleotides targeting the
human telomerase RNA component template region rep-
resent a promising approach for the treatment of HCC.
We tested two of these NPS oligonucleotides (GRN163
and GRN163L) in preclinical studies. Both compounds
signiﬁcantly inhibited in vivo tumor growth after xeno-
transplantation of two different human hepatoma cell
lines. Both cell lines possessed strong telomerase activity,
characteristic of over 80% of human HCC.
this model appears to be appropriate to study the effect of
telomerase inhibition on human hepatoma growth.
GRN163L, the lipid-conjugated derivative, inhibited tu-
mor growth more efﬁciently than GRN163, the nonlipi-
dated oligonucleotide, which is consistent with its
improved potency against tumor cells in culture and its
good biodistribution and uptake by tumor cells in vivo
following systemic administration.
The reduction in tu-
mor growth was correlated with telomerase inhibition,
induction of telomere dysfunction, decreased tumor cell
proliferation, and an increased incidence of tumor cell
apoptosis. These data are in agreement with studies in
mice showing that telomere dysfunction sup
presses tumor progression.
Tumors derived from subcutaneous engraftment of
Hep3B, Huh7, and HepG2 in nude mice have been
found to show a mild but insigniﬁcant response to doxo-
rubicin treatment (T. Wirth and S. Kubicka, unpublished
data). Interestingly, our in vivo data showed signiﬁcant
antitumor effects of telomerase inhibition alone. Our in
vitro analysis showed that GRN163L led to signiﬁcant
inhibition of telomerase activity in Hep3B, Huh7, and
HepG2 cells and, moreover, has the potential to augment
the chemosensitivity of Hep3B to doxorubicin. These re-
sults are consistent with studies showing increased sensi-
tivity of transformed mouse embryonal ﬁbroblasts from
mice compared with mTR
nal ﬁbroblasts in response to doxorubicin, and this corre-
lated with telomere shortening.
The exact mechanism
of this increased chemosensitivity has yet to be deter-
mined. Possible explanations are that functional telo-
meres are required for efﬁcient DNA repair or that agents
such as doxorubicin can accelerate the rate of telomere
loss in the absence of telomerase activity. Recent studies
have shown that mice with shortened telomeres as well as
senescent human cells have defects in DNA repair and
show an accumulation of DNA damage.
sibility is that telomerase itself fulﬁlls some function in
telomere capping and DNA repair, and that under con-
ditions in which telomerase is inhibited, genomic insta-
What mechanisms limit tumor cell growth in response
to telomerase inhibition? Studies in mTR
other studies in primary human cells have shown that
critical telomere shortening induces a DNA damage–type
response, including the activation of p53 signaling.
Table 1. Pretreatment With GRN163L Increases Hep3B
Chemosensitivity to Doxorubicin
mol/L Treatment Period
5d ⫺ 24 h 3.5
⫹ 24 h 1.3
13 d ⫺ 48 h 1.6
⫹ 48 h 0.5
14 d ⫺ 24 h 4.7
⫹ 24 h 0.9
Fig. 6. Pretreatment with GRN163L increases Hep3B chemosensitivity
to doxorubicin. GRN163L (1
mol/L) was given in the medium (Eagle
mimimal essential medium) for a period of (A) 5, (B) 13, and (C) 14
days. One day before doxorubicin treatment, fresh medium containing
GRN163L was added to the cultures. Doxorubicin (4-8 concentrations)
was added for a period of (A,C) 24 and (B) 48 hours.
1134 DJOJOSUBROTO ET AL. HEPATOLOGY, November 2005
our studies, we show that telomerase inhibition results in
increased rates of telomere-free ends and increased num-
bers of DNA damage foci in two different hepatoma cell
lines. Studies in mTR
double mutant mice
have also demonstrated evidence for p53-independent re-
sponses induced by telomere dysfunction.
In liver cells,
the level of telomere dysfunction appears to determine
whether p53-dependent or p53-independent responses
The Hep3B and Huh7 cell lines used in
this study are p53-mutant,
yet show relatively rapid and
strong inhibition of tumor growth in response to telom-
erase inhibition. These data, together with other reports,
suggest that p53 might not be a strong modulator in telo-
mere dysfunction–induced responses affecting the viabil-
ity of the hepatoma cells. It remains to be determined
what genetic alterations might inﬂuence the sensitivity of
hepatoma cells in cancer patients during telomerase inhi-
In conclusion, telomerase inhibition is a promising
therapy of human hepatoma. It has been suggested that
telomerase inhibition may only result in tumor growth
inhibition when telomeres have reached a critically short
If this were the case, the efﬁcacy of telomerase
inhibition in the clinical setting would depend on the
initial telomere length of each individual tumor. In this
regard, it is interesting that human HCC is characterized
by very short telomeres,
making this tumor type a
good target for telomerase inhibition therapy. In addi-
tion, our study showed that even the tumor-xenograft
from HCC cell lines with relatively long telomeres
(Huh7) responded quickly to GRN163L. Thus, our ex-
pectation is that most human HCC patients should re-
spond to GRN163L, either alone or in combination with
other therapeutic agents.
Given that the normal liver is telomerase-negative (re-
viewed in Lechel et al.
), telomerase inhibition should
have little effect on chronic liver disease and cirrhosis
progression. We did not notice any signiﬁcant adverse
effects of GRN163 or GRN163L in these experiments,
suggesting that these telomerase inhibitors are well toler-
ated over this treatment interval (2-4 weeks). In humans,
most somatic tissues, including normal liver, are telomer-
However, certain progenitor cells in hu-
mans are telomerase-positive; thus it remains possible that
a long-term treatment with telomerase inhibitors could
limit the proliferative potential of such progenitor cells.
Given the lack of efﬁcient therapies and the short survival
of patients with advanced HCC, a careful evaluation of
telomerase inhibitors in clinical trials appears to be a rea-
sonable approach to hopefully improve our therapeutic
option for this devastating cancer.
GRN163L has recently received clearance by the U.S.
Food and Drug Administration to enter human phase I/II
clinical testing in chronic lymphocytic leukemia. This
trial is designed to establish safety and tolerability of
GRN163L administered on a weekly intravenous dosing
schedule and to study human pharmacokinetic and phar-
macodynamic parameters. A safe starting dose and esca-
lation schedule of GRN163L considered sufﬁcient to
achieve telomerase inhibition in humans was based on in
vitro potency, in vivo efﬁcacy, and pharmacokinetic,
pharmacodynamic, biodistribution, and toxicity studies
in rodents and cynomolgus monkeys (data not shown).
We look forward to future clinical testing of GRN163L in
hematological and solid tumors.
Acknowledgment: We are grateful to A. Schienke, N.
Hadiman, and E. Wunder for technical assistance. We
thank P. Wirapati for his help in image quantitation and
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