Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo.
ABSTRACT The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo.
Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L.
Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model.
We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.
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ABSTRACT: The purpose of this review is to highlight the importance of telomeres, the mechanisms implicated in their maintenance, and their role in the etiology as well as the treatment of human esophageal cancer. We will also discuss the role of telomeres in the maintenance and preservation of genomic integrity, the consequences of telomere dysfunction, and the various factors that may affect telomere health in esophageal tissue predisposing it to oncogenesis. There has been growing evidence that telomeres, which can be affected by various intrinsic and extrinsic factors, contribute to genomic instability, oncogenesis, as well as proliferation of cancer cells. Telomeres are the protective DNA-protein complexes at chromosome ends. Telomeric DNA undergoes progressive shortening with age leading to cellular senescence and/or apoptosis. If senescence/apoptosis is prevented as a consequence of specific genomic changes, continued proliferation leads to very short (ie, dysfunctional) telomeres that can potentially cause genomic instability, thus, increasing the risk for activation of telomere maintenance mechanisms and oncogenesis. Like many other cancers, esophageal cancer cells have short telomeres and elevated telomerase, the enzyme that maintains telomeres in most cancer cells. Homologous recombination, which is implicated in the alternate pathway of telomere elongation, is also elevated in Barrett's-associated esophageal adenocarcinoma. Evidence from our laboratory indicates that both telomerase and homologous recombination contribute to telomere maintenance, DNA repair, and the ongoing survival of esophageal cancer cells. This indicates that telomere maintenance mechanisms may potentially be targeted to make esophageal cancer cells static. The rate at which telomeres in healthy cells shorten is determined by a number of intrinsic and extrinsic factors, including those associated with lifestyle. Avoidance of factors that may directly or indirectly injure esophageal tissue including its telomeric and other genomic DNA can not only reduce the risk of development of esophageal cancer but may also have positive impact on overall health and lifespan.Translational research : the journal of laboratory and clinical medicine. 09/2013;
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ABSTRACT: Telomerase is required for the unlimited lifespan of cancer cells. The vast majority of pancreatic adenocarcinomas overexpress telomerase activity and blocking telomerase could limit their lifespan. GRN163L (Imetelstat) is a lipid-conjugated N3'→P5' thio-phosphoramidate oligonucleotide that blocks the template region of telomerase. The aim of this study was to define the effects of long-term GRN163L exposure on the maintenance of telomeres and lifespan of pancreatic cancer cells. Telomere size, telomerase activity, and telomerase inhibition response to GRN163L were measured in a panel of 10 pancreatic cancer cell lines. The cell lines exhibited large differences in levels of telomerase activity (46-fold variation), but most lines had very short telomeres (2-3 kb in size). GRN163L inhibited telomerase in all 10 pancreatic cancer cell lines, with IC50 ranging from 50 nM to 200 nM. Continuous GRN163L exposure of CAPAN1 (IC50 = 75 nM) and CD18 cells (IC50 = 204 nM) resulted in an initial rapid shortening of the telomeres followed by the maintenance of extremely short but stable telomeres. Continuous exposure to the drug eventually led to crisis and to a complete loss of viability after 47 (CAPAN1) and 69 (CD18) doublings. Crisis In these cells was accompanied by activation of a DNA damage response (γ-H2AX) and evidence of both senescence (SA-β-galactosidase activity) and apoptosis (sub-G1 DNA content, PARP cleavage). Removal of the drug after long-term GRN163L exposure led to a reactivation of telomerase and re-elongation of telomeres in the third week of cultivation without GRN163L. These findings show that the lifespan of pancreatic cancer cells can be limited by continuous telomerase inhibition. These results should facilitate the design of future clinical trials of GRN163L in patients with pancreatic cancer.PLoS ONE 01/2014; 9(1):e85155. · 3.53 Impact Factor
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ABSTRACT: Telomere shortening is observed in peripheral mononuclear cells from patients with major depressive disorder (MDD). Whether this finding and its biological causes impact the health of the brain in MDD is unknown. Brain cells have differing vulnerabilities to biological mechanisms known to play a role in accelerating telomere shortening. Here, two glia cell populations (oligodendrocytes and astrocytes) known to have different vulnerabilities to a key mediator of telomere shortening, oxidative stress, were studied. The two cell populations were separately collected by laser capture micro-dissection of two white matter regions shown previously to demonstrate pathology in MDD patients. Cells were collected from brain donors with MDD at the time of death and age-matched psychiatrically normal control donors (N = 12 donor pairs). Relative telomere lengths in white matter oligodendrocytes, but not astrocytes, from both brain regions were significantly shorter for MDD donors as compared to matched control donors. Gene expression levels of telomerase reverse transcriptase were significantly lower in white matter oligodendrocytes from MDD as compared to control donors. Likewise, the gene expression of oxidative defence enzymes superoxide dismutases (SOD1 and SOD2), catalase (CAT) and glutathione peroxidase (GPX1) were significantly lower in oligodendrocytes from MDD as compared to control donors. No such gene expression changes were observed in astrocytes from MDD donors. These findings suggest that attenuated oxidative stress defence and deficient telomerase contribute to telomere shortening in oligodendrocytes in MDD, and suggest an aetiological link between telomere shortening and white matter abnormalities previously described in MDD.06/2014;
Telomere Maintenance in Laser Capture Microdissection^Purified
Barrett’s Adenocarcinoma Cells and Effect of Telomerase
Inhibition In vivo
Masood A. Shammas,1Aamer Qazi,1Ramesh B. Batchu,1Robert C. Bertheau,2,3,4Jason Y.Y. Wong,3,5
Manjula Y. Rao,3,4Madhu Prasad,1Diptiman Chanda,6Selvarangan Ponnazhagan,6
Kenneth C. Anderson,2,3Christopher P. Steffes,1Nikhil C. Munshi,2,3,4
Immaculata De Vivo,3,5David G. Beer,7Sergei Gryaznov,8
Donald W. Weaver,1and Raj K. Goyal3,4
Purpose:The aims of this study were to investigate telomere function in normal and Barrett’s
esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to
evaluate the effectof telomeraseinhibitionin cancer cells invitro and invivo.
Experimental Design: Epithelial cells were purified from surgically resected esophagi. Telo-
merase activity was measured by modified telomeric repeat amplification protocol and telomere
length was determined by real-time PCR assay.To evaluate the effect of telomerase inhibition,
adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor
(GRN163L) and live cell number was determined weekly. Apoptosis was evaluated byAnnexin
labeling and senescence by h-galactosidase staining. For in vivo studies, severe combined
immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance
of palpable tumors, injectedi.p. with saline or GRN163L.
Results:Telomerase activity was significantlyelevated whereas telomeres were shorterin BEAC
cells relative to normal esophageal epithelial cells.The treatment of adenocarcinoma cells with
telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length,
and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced
cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and
ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a
subcutaneous tumor model.
Conclusions: We show that telomerase activity is significantly elevated whereas telomeres
are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells
both invitro and invivo.
The ends of eukaryotic chromosomes associate with specific
DNA binding proteins to form specialized nucleoprotein
structures that play a vital role in maintaining genomic integrity
by preventing exonucleolytic degradation and end-to-end
fusion of chromosomal DNA (1). In humans, the telomeric
DNA is composed of few hundred to several thousand repeats
of a conserved DNA sequence ‘‘TTAGGG’’ (2–4), which is asso-
ciated with at least six proteins, TRF1, TRF2, TIN2, HRAP,
POT1, and TPP (5), each with a specific role in the maintenance
of telomeric DNA length and genomic integrity of a cell.
Telomere length in normal somatic cells shortens with each
cell division. Because DNA polymerases require a RNA primer
to initiate DNA synthesis, they are unable to replicate the 5¶ end
of lagging strand of DNA, leading to a loss of 50 to 100 bp of
telomeric DNA with each cell division in normal human cells
(6). When telomere length reaches below the critical limit of
2 kb, the critically short telomeres are recognized as DNA
damage and may lead to either apoptosis and/or cellular
senescence (7–9). The length of telomeric DNA may therefore
act as a mitotic clock (10), which determines the life span of
normal human somatic cells. Although telomeres play a vital
Cancer Therapy: Preclinical
Authors’Affiliations:1Department of Surgery, Wayne State University and
Karmanos Cancer Institute, Detroit, Michigan;2Dana-Farber Cancer Institute,
3Harvard Medical School,4VA Boston Healthcare System, and5Department of
Medicine, Brigham andWomen’s Hospital, Boston, Massachusetts;6University of
Alabama at Birmingham, Birmingham, Alabama;7University of Michigan, Ann
Arbor, Michigan, and8Geron, Corporation, Menlo Park, California
Received 2/20/08; revised 3/27/08; accepted 3/30/08.
Grant support: NIH-P50-100007 Developmental Research Award (M.A.
Shammas); Department ofVeterans Affairs Merit ReviewAwards Research Service
and Department ofVeterans Affairs Merit ReviewAwards Epidemiology Service;
NIH grants P50-100007 and PO1-78378 (N.C. Munshi); and Karmanos Cancer
Institute, Detroit, Michigan.
The costs of publicationof this articlewere defrayedinpart by the paymentof page
charges.This article must therefore be hereby marked advertisement in accordance
with18 U.S.C. Section1734 solely toindicatethis fact.
Note: M.A. Shammas, A. Qazi, and R.B. Batchu contributedequally to this work.
Requests for reprints: Raj K. Goyal, Harvard Medical School atVeterans Affairs
Medical Center,1400 VFW Parkway,West Roxbury, MA 02132. Phone: 857-203-
5612; E-mail: Raj___goyal@hms.harvard.edu or Masood A. Shammas, Karmanos
Cancer Institute/Wayne State University, 615, HudsonWebber Cancer Research
Center, 4100 John R. Street, Detroit, MI 48201. Phone: 313-576-8869; E-mail:
F2008 American Association for Cancer Research.
www.aacrjournals.orgClin Cancer Res 2008;14(15) August1, 20084971
role in the maintenance of genomic integrity and cellular health
(11–14), telomere dysfunction or excessive telomere shorten-
ing caused by mutation or inherited disorder may result in
genetic instability (15) and development of cancer (16).
Consistent with these findings, it has been shown that telomere
length may serve as a marker for progression and/or prognosis
for cancers such as neuroblastoma (17), prostate, colon, breast,
brain, head and neck, and lung (18).
Telomere length is maintained by telomerase, an enzyme
with a RNA component (TERC) containing a short template
for the synthesis of TTAGGG telomere repeats (19) and a
polypeptide component with a reverse transcriptase activity
(TERT). Telomerase extends telomere length by adding
TTAGGG sequences to guanine-rich strand of telomeric DNA.
The activity of telomerase depends on posttranslational and
posttranscriptional modifications of the protein component
(hTERT), including phosphorylation, assembly into telomerase
holoenzyme, and its association with other proteins such as
p23, and hsp90, etc. (6, 20). Telomerase activity is present in
human germ-line cells that maintain their telomere length (21).
Conversely, telomerase activity is low or undetectable in normal
somatic tissues in which telomeres are not extended and,
therefore, undergo progressive shortening with cell division
(22). Telomerase knockout mice show a number of abnormal-
ities, including hair graying, alopecia, higher incidence of skin
lesions, reduced body weight, delayed healing, diminished
hematopoietic reserve, and atrophy of small intestinal cells by
generation 3 (23). Additionally, they show hypoprolifera-
tive defects in lymphoid, hematopoietic, and gonadal cells by
generation 6 (24). Conversely, the induction of telomerase
in normal human cells increases their life span in culture (25)
and oncogenic conversion in the presence of SV40 T antigen and
H-ras (26). Consistent with this, telomerase is reactivated in
most immortalized cells (27–31) and cancers (30).
Although a majority of immortal and cancer cells have
elevated telomerase activity, a subset of immortal and cancer
cell lines lack detectable telomerase activity, and maintain their
telomeres through an alternative mechanism (32). Consistent
with this, Opitz et al. (33) have shown the immortalization of
human primary oral squamous epithelial cells through a
mechanism independent of telomerase. However, it has also
been shown that human cells can use both telomere main-
tenance mechanisms, telomerase and an alternative mecha-
nism, at the same time (34).
In cancer cells, telomerase activity is elevated and telomeres
are shorter but stable (35, 36). Because telomeres are shorter in
cancer cells relative to normal cells whereas telomerase activity
is elevated in most cancers but absent or low in normal somatic
cells (21, 29, 37), the inhibitors of telomerase activity have a
strong potential to be used as anticancer therapeutics, which
may inhibit proliferation of tumor cells while having little or
no effect on normal cells. Low levels of telomerase activity is
also detected in some normal somatic cells such as hemato-
poietic, peripheral blood, and gastroesophageal cells; however,
transient telomerase inhibition may not affect them as
telomeres in normal cells are significantly longer than those
in cancer. Consistent with this, we have shown that treatment
of various cancer (8, 9, 38–40) and immortal (human cell
lines; refs. 41, 42) cell lines with a variety of telomerase
inhibitors results in the loss of telomerase activity, telomere
shortening, and reversal of immortality.
Morales et al. (43) have shown that the expression of RNA
component of telomerase (hTR) is increased in surgical
specimens of Barrett’s esophagus and further elevated in high-
grade dysplasia and esophageal adenocarcinoma. Consistent
with this report, studies by Lord et al. (44) indicate that
transcript levels of catalytic subunit of telomerase (hTERT) are
also elevated early at Barrett’s esophagus stage with a further
up-regulation in specimens of high-grade dysplasia and
adenocarcinoma. These studies may indicate that telomerase
expression is increased early at Barrett’s esophagus stage;
however, levels of hTR and hTERT do not reflect telomerase
activity, the ability of enzyme to add telomeric repeats.
Although hTERT mRNA expression correlates with telomerase
activity in many cancers, it does not measure telomerase activity
(45), which depends on posttranscriptional and posttransla-
tional modifications of hTERT, including phosphorylation of
hTERT protein, assembly into telomerase holoenzyme, and
its association with other proteins such as hsp90 and p23, etc.
The aims of this study were to (a) assess telomerase activity
and telomere length in primary human cells [normal and
cancer; Barrett’s esophageal adenocarcinoma (BEAC)] purified
by laser capture microdissection and (b) evaluate the efficacy of
a specific telomerase inhibitor, GRN163L, in vitro and in vivo.
We show that BEAC cells purified from surgical specimens
by laser capture microdissection (LCM) have significantly
elevated telomerase activity and markedly reduced telomeres
relative to normal esophageal epithelial cells. The treatment of
adenocarcinoma cells with a specific and potent telomerase
inhibitor, GRN163L, led to loss of telomerase activity, further
reduction in telomere length, and inhibition of cell growth
through induction of both the senescence and apoptosis.
GRN163L-induced cell death could also be expedited by
combination treatment with doxorubicin (a DNA-interacting
drug) or ritonavir (a protease inhibitor). Addition of these
drugs to cultures pretreated with GRN163L had a significant
additive effect on GRN163L-induced cancer cell death. Efficacy
of GRN163L was also tested in a murine model in which severe
combined immunodeficient mice (SCID) mice were s.c.
inoculated with SEG-1 adenocarcinoma cells and, following
appearance of palpable tumors, treated with either PBS or
GRN163L. A significant reduction in tumor volume was seen in
mice treated with the drug.
Materials and Methods
and frozen at -80jC from surgically resected esophagi bearing various
Barrett’s esophagus–related lesions, including adenocarcinoma, were
used for this study. The protocol for this study is already approved by
the institutional review board of the University of Michigan, Ann Arbor,
MI. The tissue specimens were coded without use of any of patient
private identifiers. For all specimens, only the mucosa containing the
columnar epithelium or cancer or normal squamous mucosa was
selected by macrodissection. The presence of goblet cells was diagnostic
for Barrett’s mucosa. The specimens of five normal, one Barrett’s, and
five BEAC were used for this study and the Barrett’s esophagus specimen
was from the patient who had cancer. Populations of normal and
abnormal cells were isolated by LCM.
Acquisition of target cells with LCM.
tissue sections were obtained using the LCM technique as follows:
Frozen tissue sections (8 Am) were cut, mounted onto glass slides, fixed
Tissue samples that were immediately processed
Target cells from fresh-frozen
Cancer Therapy: Preclinical
www.aacrjournals.orgClin Cancer Res 2008;14(15) August1, 20084972
(70% ethanol for 3 min), and stained with hematoxylin. The stained
sections were examined under a microscope by an experienced
gastrointestinal pathologist and the targeted area, including various
Barrett’s esophagus–related lesions, was identified according to the
standard histopathologic criteria (46). Specialized intestinal metaplasia
was defined as specialized columnar epithelium containing well-
formed goblet cells whereas BEAC was defined with the breakdown
of the basement membrane by dysplastic glands that present within the
laminar propria and beyond. Once the targeted glands were identified,
the area was marked with a marker and the slide was repositioned for
LCM using the Pixcell II LCM System (Arcturus, Inc.), according to the
standard protocol (47). The captured cells were placed in an
appropriate buffer for subsequent analysis.
Telomere length analysis.Genomic DNA was isolated from captured
cells using the QIAamp DNA Micro Kit (Qiagen). Average relative
telomere length as represented by the telomere repeat copy number to
single copy gene copy number (T/S) ratio was determined using a
modified version of a previously described real-time PCR assay on an
Applied Biosystems 7900HT Thermocycler (48). Briefly, 5 ng of
genomic DNA were dried down in a 384-well plate and resuspended
in 10 AL of either the telomere or 36B4 quantitative PCR reaction
mixture. Primer sequences of Tel-1, Tel-2, 36B4-U, and 36B4-D were
previously described (48). The telomere reaction mixture consisted of
1? Qiagen Quantitect Sybr Green Master Mix, 2.5 mmol/L of DTT,
270 nmol/L of Tel-1, and 900 nmol/L of Tel-2 primer. The reaction
proceeded for 1 cycle at 95jC for 5 min, followed by 40 cycles at 95jC
for 15 sec and at 54jC for 2 min. The 36B4 reaction consisted of
1? Qiagen Quantitect Sybr Green Master Mix, 300 nmol/L of 36B4U
primer, and 500 nmol/L of 36B4D primer. The 36B4 reaction
proceeded for one cycle at 95jC for 5 min, followed by 40 cycles at
95jC for 15 sec and at 58jC for 1 min. All samples for both the
telomere and 36B4 reactions were done in triplicate. In addition to the
samples, each 384-well plate contained a 6-point standard curve from
1.25 to 30 ng using pooled buffy coat–derived genomic DNA. The
slope of the standard curve for both the telomere and 36B4 reactions
was -3.6 F 0.2 and the linear correlation coefficient (R2) value for both
reactions was 0.98 and 0.99, respectively. The T/S ratio for each sample
was calculated by subtracting the mean 36B4 Ct value from the mean
telomere Ct value. The T/S ratio values were then linearized to 2-(-dCt).
Assay of telomerase activity.For telomerase activity assays, the cells
were immediately lysed in CHAPS lysis buffer (Chemicon International,
Inc.) at 50 cells/AL and stored at -150jC. Telomerase activity was
measured by an improved version of the original telomeric repeat
amplification protocol, using the TRAPeze XL Telomerase Detection Kit
(Chemicon International), as previously reported (8, 9, 39). TRAPeze
XL is a fluorescence-based highly sensitive assay and provides
quantitative analysis of telomerase activity. Using this assay, telomerase
activity can be detected in extracts from as few as 100 telomerase-
positive cells. In triplicate, cell lysates were mixed with TRAPeze XL
reaction mix containing Amplifuor primers and incubated at 30jC for
30 min. Amplified telomerase products were quantitated with FLUOstar
OPTIMA Fluorescence Plate Reader (BMG LABTECH). Telomerase
activity (in TPG units) was then calculated by comparing the ratio of
telomerase products to an internal standard for each lysate, as described
by the manufacturer. The assay has a linear range of 1 to 300 TPG units,
equivalent to telomerase activity from f30 to 10,000 control cells.
Telomerase inhibitor GRN163L.
lipid-attached oligonucleotide (5¶-Palm-TAGGGTTAGACAA 3¶) target-
ing the RNA component of telomerase and having a N3¶-P5¶-thio-
Cell lines and treatments.Adenocarcinoma cell lines BIC-1, SEG-1,
and FLO-1 have been described previously (49). Cells were cultured in
DMEM with 10% fetal bovine serum, as described previously (49).
Constant numbers of cells were plated in multiple 100-mm dishes;
treated with GRN163L at 0.5 to 2.0 mmol/L; and evaluated weekly for
cell viability, telomere length, and telomerase activity. Briefly, every
week, the cells were harvested and counted, and the viable cell number
GRN163L is a palmitoyl (C16)
was confirmed by trypan blue exclusion and 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Aliquots of cells
were separated for various molecular analyses and the remaining cells
were replated at the same cell number and at the same concentration of
the inhibitor. For combination studies, cells were pretreated with
GRN163L for 10 d. Test agents were then added at various concen-
trations and live cell number was determined at alternate days.
Apoptosis.Following exposure to GRN163L, apoptotic cells were
detected by Annexin labeling using FITC-Annexin Apoptosis Detection
Kit (Oncogene Research Products). Briefly, the cells treated with
GRN163L were harvested and 0.5 mL of cells (1 ? 106/mL) was mixed
with FITC-Annexin in ‘‘binding buffer’’ and incubated for 15 min at
room temperature. A portion of cell suspension (50 AL) was placed onto
a glass slide, covered with a coverslip, and viewed immediately using
a fluorescence microscope equipped with FITC (green) filter. Approxi-
analyzed to assess the fraction of FITC-labeled cells for each sample.
Senescence. One day before this assay, the treated cells were plated
on Lab-Tek slides in the presence of mismatch or match (GRN163L)
oligonucleotide and the attached cells were stained for h-galactosidase
expression, a marker for cellular senescence (50). Briefly, the cells were
rinsed thrice with PBS and fixed in 2% formaldehyde and 0.2%
glutaraldehyde solution in PBS. The cells were then washed again as
described above and stained overnight in solution containing 1 mg/mL
X-gal, 40 mmol/L citric acid/sodium phosphate (pH 6), 5 mmol/L
potassium ferrocyanide, 150 mmol/L NaCl, and 2 mmol/L MgCl2. The
stain was then removed, cells were rinsed with PBS, and staining was
viewed under a fluorescence microscope (Olympus).
Murine subcutaneous tumor model.
the National Cancer Institute-Frederick Animal Production area.
Maintenance of the animals was carried out following guidelines of
the Institutional Animal Care and Use Committee and all experimental
procedures were approved by the Institutional Animal Care and Use
Committee and the Occupational Health and Safety Department of the
University of Alabama at Birmingham. The mice were acclimatized for a
week and 3.0 ? 106SEG-1 cells in 100 AL saline were injected s.c. in the
interscapular area. Following appearance of palpable tumors, mice were
injected i.p. with normal saline alone or GRN163L at concentrations
described in the figure legends. Animals were sacrificed when tumors
reached 2 mL in volume or when paralysis or major compromise in
their quality of life occurred.
SCID mice were purchased from
LCM significantly enhances the sensitivity of telomerase activity
assay. Epithelial cells were purified from tissue specimens of
normal and Barrett’s esophagi as shown in Fig. 1A, and
telomerase activity was measured in these purified cells and
also in tissue extracts of the same specimens, using same
amount of total protein. Telomerase activity in tissue extracts of
normal and Barrett’s esophagus was only 3.1 F 0.9 and 19 F
4.6 TPG units, respectively. However, the activity in LCM-
purified normal and Barrett’s esophageal cells was 37.7 F
5.5 and 197 F 23.1 TPG units, respectively (Fig. 1B). These data
show that telomerase activity is f10-fold higher in LCM-
derived cells relative to that in tissue extracts of the same
samples. To investigate if the low activity detected in tissue
extracts was due to PCR inhibitors, both the tissue extracts and
lysates of LCM-purified Barrett’s esophagus cells were diluted
and evaluated for telomerase activity. A 10-fold dilution of
tissue extract and the lysate of LCM-purified cells of Barrett’s
esophagus led to 11.4- and 9.2-fold reduction in telomerase
Telomerase activity is markedly elevated in BEAC relative to
normal esophageal epithelial cells. Epithelial cells purified from
Telomeres,Telomerase, and Barrett’s Adenocarcinoma
www.aacrjournals.orgClin Cancer Res 2008;14(15) August1, 20084973
tissue specimens of normal, Barrett’s adenocarcinoma (BEAC),
and commercially obtained primary normal esophageal epi-
thelial cells (HEEC; ScienCell) were lysed at the same cell
concentration and evaluated for telomerase activity using
TRAPeze XL Telomerase Detection Kit (Chemicon Interna-
tional). The telomerase activity in LCM-purified normal
esophageal epithelial cells from three different donors was
41 F 8 TPG units; consistent with this, the activity in com-
mercially obtained primary normal esophageal epithelial cells
(HEEC) was also low (37 F 3 TPG units; Fig. 1C). A markedly
elevated telomerase activity was detected in BEAC (275 F 20
TPG units; Fig. 1C). Relative to normal esophageal epithelial
cells, the activity in BEAC was 7.0 F 0.5-fold elevated (P =
0.004). These data indicate that telomerase activity is signifi-
cantly elevated in primary BEAC cells relative to normal
esophageal epithelial cells.
Telomeres are consistently shorter in BEAC relative to normal
esophageal epithelial cells. We next analyzed telomere length
in a panel of LCM-purified normal and BEAC cells. Genomic
DNA was isolated using QIAamp DNA Micro Kit (Qiagen) and
telomere length was measured by real-time kinetic quantitative
PCR (48). Briefly, the PCR reactions for telomere specific (T)
and h2-globin control gene specific (S) amplification were set
up in separate 96-well plates. Except primers, other conditions,
including reagent concentrations, DNA amount, and sample
order, were the same for both plates. To monitor the efficiency
of PCR reactions, a standard curve spanning at least 0.3 to
5 ng/AL of reference DNA was produced in each 96-well plate.
DNA from each sample and reference DNA were tested in
triplicate using a total of 12.5 ng DNA per well. PCR ampli-
fication reactions were done in a PRISM 7900 Sequence Detec-
tion System (Applied Biosystems) using conditions described
by Cawthon (48). Analysis of data and determination of
telomere length were done using a software (Applied Bio-
systems), as described (48). The ratio of telomere-specific (T)
and single control gene–specific (S) real-time kinetic PCR
reactions (T/S ratio) indicated the relative telomere length. The
T/S ratio, indicating relative telomere length, ranged from 10.3
to 14.3 in normal cells and 6.2 to 9.2 in BEAC cells, indicating
that telomeres in all BEAC specimens were shorter than those
in normal samples (Fig. 2A). Average TL was reduced by 38%
(P = 0.001) in BEAC relative to the length in normal epithelial
cells (Fig. 2B). These results provided a further rationale for
investigating the importance of telomerase as a target for the
treatment of BEAC.
GRN163L is taken up by adenocarcinoma cells without
transfection and inhibits telomerase activity. GRN163L is a
palmitoyl (C16) lipid-conjugated oligonucleotide N3¶-P5¶-thio-
phosphoramidate targeting template region of RNA component
of telomerase (hTR; Fig. 3A). We treated SEG-1 cells with
Fig.1. Telomerase activity in tissue extracts and esophageal epithelial cells isolated by LCM. A, isolation of esophageal epithelial cells by LCM. I, epithelial cells in a
microscopic field before LCM; II, the same microscopic field after LCM, showing that epithelial cells have been removed; III, the captured epithelial cells. B, telomerase
activity in tissue extract versus LCM-purified cells.Three normal and three BEAC surgical specimens were processed for evaluation of telomerase activity in LCMand
tissue extracts. Each surgical specimen was cut into two portions; one was processed for LCM purification of epithelial cells and the other was used for making the tissue
extract.Telomerase activity is shownin the tissue extract, equivalent of 0.6 Ag protein, of normal esophagus (lane1); tissue extract, equivalent of 0.6 Ag protein, of Barrett’s
esophagus (lane 2); dilutedtissue extract, equivalentof 0.06 Agprotein, of Barrett’s esophagus (lane3); lysate of LCM-purifiednormalesophagealepithelialcells, equivalent
of 0.6 Ag protein (lane 4); lysate of LCM-purified Barrett’s esophagus cells, equivalent of 0.6 Ag protein (lane 5); and diluted lysate, equivalent of 0.06 Ag protein, of
LCM-purified Barrett’s esophagus cells (lane 6). C, telomerase activity in defined primary normal and Barrett’s adenocarcinoma (BEAC) cells derived by LCM.The activity
wasmeasuredinthelysates (equivalentof0.6Agprotein) ofnormalprimaryesophagealepithelialcellspurchasedfromScienCell (HEEC), epithelialcellspurifiedfrom surgical
specimens of normal esophagus from three different patients, and epithelial cells purified from surgical specimens of BEAC from three different patients, using LCM.
Cancer Therapy: Preclinical
www.aacrjournals.orgClin Cancer Res 2008;14(15) August1, 20084974
TAMRA-labeled GRN163L at various concentrations and
monitored the uptake and intracellular localization at 24 hours
using a confocal microscope. As seen by TAMRA (red)
fluorescence in Fig. 3B, the drug was efficiently taken by SEG-
1 cells without any need of a transfection procedure or reagent.
The fluorescence was predominantly nuclear. An efficient
uptake of GRN163L without any need for transfection reagents
was also observed for other adenocarcinoma cell lines BIC-1
and FLO-1 (not shown).
We next evaluated if the uptake of GRN163L was also asso-
ciated with the inhibition of telomerase activity. We therefore
treated SEG-1, FLO-1, and BIC-1 cells with the drug at 0.1,
0.5, 1.0, and 2.0 Amol/L concentrations for 24 hours and
evaluated for telomerase activity using TRAPeze XL Telo-
merase Detection Kit (Intergen). GRN163L at 1 and 2 Amol/L
led to near-complete inhibition of telomerase activity in
SEG-1 cells (Fig. 3C). Mismatch control oligonucleotide thio-
phosphoramidate 5¶-Palm-TAGGTGTAAGCAA (mismatch
bases are underlined, designated as GRN140833) had no effect
on growth of these cells at either concentration. Similarly, the
treatment with 1 Amol/L of GRN163L led to >95% inhibi-
tion of telomerase activity in FLO-1 (Fig. 3D) and BIC-1 cells
(not shown). No significant inhibition of telomerase activity
was seen at 0.1 Amol/L concentration in any cell line.
Inhibition of telomerase by GRN163L leads to induction of
growth arrest in adenocarcinoma cells. Cells were treated with
GRN163L or mismatch oligonucleotide GRN140833 and the
substrate-attached viable cell number was counted every week.
Cell viability was further confirmed by trypan blue exclusion
and/or MTT assays. A marked arrest of cell proliferation was
observed in all cell lines following treatment with the inhibitor,
leading to a gradual decline in viable cell number by 79% to
82% in a span of 2 to 3 weeks. Treatment of SEG-1 cells with
the drug at 1 and 2 Amol/L concentrations induced 45% and
80% cell death, respectively, in a period of 21 days (Fig. 4A).
Similar induction of cell death was observed following 3-week
Fig. 2. Telomere lengthin esophageal epithelial cells isolated fromnormal and
Barrett’s adenocarcinoma specimens by LCM. Normal epithelial and BEAC cells
were isolated by LCM, genomic DNAwas extracted, and telomere length was
determined by quantitative PCR. A, relative telomere lengthin primary normal and
BEAC cells purified by LCM. B, average telomere lengthin five normal and five
BEAC specimens is shown as percent of telomere lengthinnormal cells.
Fig. 3. GRN163L inhibits telomerase
activity in adenocarcinoma cells.
A, structure of GRN163L, a palmitoyl (C16)
RNA component of telomerase. B, uptake
of transfection. Cells were treated with
TAMRA-labeled GRN163L for 24 h and
examined by a multiphoton fluorescence
microscope. Uptake can be seen as red
fluorescence. C and D, telomerase activity
in cells treated with a mismatch control
oligonucleotide (MM) or GRN163L. SEG-1
(C) and FLO-1 (D) were treated with
GRN163L at various concentrations for
24 h and evaluated for telomerase activity
usingTRAPeze XLTelomerase Detection Kit.
Telomeres,Telomerase, and Barrett’s Adenocarcinoma
www.aacrjournals.org Clin Cancer Res 2008;14(15) August1, 20084975
treatments of FLO-1 (Fig. 4B) and BIC-1 (not shown).
Consistent with telomerase activity data, 1 Amol/L GRN163L
was enough to induce a marked (80%) growth inhibition in
FLO-1 cells (Fig. 4B).
GRN163L-induced inhibition of telomerase activity is asso-
ciated with reduction in telomere length. We also analyzed
telomere length following exposure of cells to GRN163L using
the same real-time kinetic quantitative PCR as described above
(48). Telomere length in GRN163L-treated SEG-1 and FLO-1
cells was reduced by 38% and 67%, respectively, relative to cells
treated with mismatch control oligonucleotide (Fig. 4C and D).
These data indicate that inhibition of telomerase and growth
arrest following treatment with GRN163 was also associated
with reduction in telomere length.
The nature of cell death following GRN163L treatment. Cell
lines were treated with 1 Amol/L GRN163L and evaluated
for apoptosis and senescence. Apoptotic cells were detected
using an Annexin V-FITC Apoptosis Detection Kit (BD
Biosciences). Following 2 weeks of treatment with 2 Amol/L
GRN163L, 85 F 9% of SEG-1 cells stained positive for Annexin
V, whereas only 2.4 F 2% Annexin V–positive cells were
detected when the cells were treated with mismatch oligonu-
cleotide (Fig. 5A). Annexin labeling was also detected in a
majority (57 F 3%) of FLO-1 cells treated with drug (Fig. 5B).
These data indicate that telomerase inhibition and telomere
shortening were associated with induction of apoptosis in
GRN163L-treated cells were also evaluated for expression of
h-galactosidase, a marker for cell senescence. Interestingly, a
large fraction (64 F 7%) of SEG-1 cells treated with GRN163L
also stained positive for h-galactosidase. Moreover, a subset of
GRN163L-treated cells also showed typical senescent morphol-
ogy and became large in size. Such cells, indicated with red
arrows, could be seen in SEG-1 (Fig. 5C), FLO-1 (Fig. 5D), and
BIC-1 cells (not shown). These data indicate that GRN163L
induced both the senescence and apoptosis in adenocarci-
Effect of combining GRN163L-mediated telomerase inhibition
with other agents. We have also evaluated the effect of other
novel agents on GRN163L-induced adenocarcinoma cell death.
Of several agents tested, a DNA-interacting drug ‘‘doxorubicin’’
and a protease inhibitor ‘‘ritonavir’’ had a significant additive
effect on GRN163L-induced cell death. Addition of ritonavir
and doxorubicin to cultures pretreated with GRN163L for
10 days led to z80% cell death within 3 days of addition
(Fig. 6A), compared with significantly higher viability of cells
treated with either drug alone. These data indicate that cancer
cell death following inhibition of telomerase can also be
expedited by addition of other agents that may affect telomere
length by other mechanisms.
Fig. 4. Effectof GRN163Longrowthandtelomerelengthofadenocarcinoma cells. A and B, cells were culturedinregulargrowthmedium containing GRN163Lormismatch
control oligonucleotide at concentrations shown and live cellnumber was determined at different time points as indicated. Points, mean of three independent experiments;
bars,SE.A, SEG-1cells; B, FLO-1cells; C and D, telomere shorteningincells treatedwith GRN163L. Cells were treatedwithmismatch controloligonucleotide or GRN163L at
1Amol/Lfor3wkandtelomerelengthwasexaminedbyquantitativePCR.C, relativetelomerelengthincontrolandGRN163L-treatedSEG-1cells.D, relativetelomerelengthin
control and GRN163L-treated FLO-1cells.
Cancer Therapy: Preclinical
www.aacrjournals.org Clin Cancer Res 2008;14(15) August1, 20084976
Telomerase inhibitor GRN163L inhibits adenocarcinoma cell
growth in vivo. The efficacy of GRN163L was shown in a
subcutaneous tumor model. Briefly, the SCID mice were
s.c. inoculated in the interscapular area with 3.0 ? 106SEG-1
cells and, following appearance of palpable tumors, mice
were injected i.p. with saline alone or GRN163L at 45 mg/kg/d.
The tumor size in mice treated with 163L was significantly
smaller than control (average tumor size in treated mice
being >10-fold smaller; P = 0.03), indicating a marked efficacy
In this article, we studied telomere maintenance in primary
human BEAC and normal esophageal epithelial cells isolated
by LCM and evaluated the effect of telomerase inhibition in
Fig. 5. Apoptosis and senescence in adenocarcinoma cells treated with GRN163L. A and B, apoptotic cell deathin cells treated with GRN163L.The cells treated with
control oligonucleotide or GRN163L were harvested; 0.5 mL of cells (1?106/mL) were mixed with FITC-Annexin andincubated for15 min at room temperature. A portion
of cell suspension (50 AL) was placed onto a glass slide covered with a coverslip, and FITC-labeled apoptotic cells within the same microscopic field were viewed and
photographedby phase contrast or by fluorescence emitted at 518 nm (FITC filter). Apoptotic cells appear bright green. A, SEG-1cells treated with 2 Amol/L control
oligonucleotide or GRN163L for 3 wk. B, FLO-1cells, treated with1 Amol/L control oligonucleotide or GRN163L for 2 wk. Approximately 200 to 300 cells representing
five differentmicroscopic fieldswere evaluatedto assess percentage ofapoptotic cells, shownasbar graphs in A and B.C and D, cells treatedwith GRN163Lwere evaluated
for expression of h-galactosidase, a marker of cell senescence. C, h-galactosidase staining is shownin SEG-1cells treated with 2 Amol/L control oligonucleotide (I) or
GRN163L (II) for 3 wk. III, bar graph showing the percentage of senescent SEG-1cells. D, FLO-1cells treated with1 Amol/L control oligonucleotide (I) or GRN163L (II) for
2 wk. Arrows (C and D), cells with typical senescent morphology.
Telomeres,Telomerase, and Barrett’s Adenocarcinoma
www.aacrjournals.orgClin Cancer Res 2008;14(15) August1, 2008 4977
three different adenocarcinoma cell lines and in a subcutaneous
tumor model using SCID mice. We have shown that (a)
telomerase activity assays conducted in tissue extracts can
produce inaccurate results whereas the assays conducted in
LCM-purified specimens are more accurate and reliable; (b)
telomerase activity is significantly elevated whereas telomeres
are significantly shorter in BEAC relative to normal esophageal
epithelial cells; (c) GRN163L, a lipid -conjugated oligonucle-
otide telomerase template antagonist, is efficiently taken up by
adenocarcinoma cells without any transfection and leads to
inhibition of telomerase activity within 24 hours; (d) inhibi-
tion of telomerase activity is associated with growth arrest,
reduction in telomere length, and induction of both the
senescence and apoptosis; (e) a significant additive effect of
doxorubicin and ritonavir was observed on GRN163L-induced
growth inhibition; and (f) i.p. injections of GRN163L caused a
significant reduction in tumor size in vivo.
A large discrepancy has been reported for telomerase activity
in normal and Barrett’s esophagi (51–54) and for the activity
observed in surgical and endoscopic biopsy specimens (51).
The major cause of this controversy is the fact that these studies
have been conducted on tissue extracts contaminated with
varying proportions of connective tissue cells. These contam-
inating cells do not only dilute the lysate but may also have an
inhibitory effect on telomerase activity. Bachor et al. (51) have
also proposed that the discrepancy arises because of contam-
inating connective tissue, which is more in surgically obtained
specimens and less in superficial biopsies. Consistent with this,
it has been shown that removal of dermis from epidermis led to
an easier detection of telomerase activity in human skin (55).
In an attempt to resolve the discrepancy, we have evaluated and
compared telomerase activity in tissue extracts and LCM-
purified primary epithelial cells from normal and Barrett’s
esophagi. As shown in Fig. 1, the telomerase activity was
f10-fold higher in LCM-derived cells relative to that in tissue
extracts of the same samples, containing the same amount of
protein. The most likely explanation of this observation is that
the activity in tissue extracts is substantially diluted because of
the contaminating (connective tissue, blood vessels, nerve
fibers, and smooth muscle) cells in mucosal tissue. Alterna-
tively, the low telomerase activity detected in tissue extracts
could also be due to the presence of PCR inhibitors. However, if
this is true, the dilution of the samples should lead to an
increase in activity. We therefore diluted both the tissue extracts
and LCM samples and evaluated them for telomerase activity.
The dilution did not increase; however, decreased telomerase
activity and the extent of decline were consistent with the linear
standard curve generated from dilutions of a control template.
It is therefore less likely that the low telomerase activity
detected in tissue extracts in these experiments was due to the
presence of PCR inhibitors. We therefore conclude that telo-
merase assays conducted in tissue extracts, containing varying
proportions of contaminating cells, may produce inaccurate
results because of the dilution effect. Purification of the specific
cells under investigation can avoid all these problems and
variables. We would also like to caution that the data shown in
Fig. 1B indicate the importance of LCM purification but may not
confirm that telomerase activity is elevated in Barrett’s esophagi,
in general, because of insufficient sample size.
length in laser captured cells. Normal squamous epithelial cells
were used as control for BEAC. It has been debatable as to what
the appropriate control cells are for Barrett’s esophagus.
Although Barrett’s esophagus cells are columnar, they do not
arise from either gastric or intestinal cells. We elected normal
squamous epithelial cells as controls and found that telomerase
activity is significantly elevated in BEAC. This is consistent with
earlier reports of increased telomerase RNA (43) and catalytic
with other agents andin a subcutaneous
tumor model. A, SEG-1cells were
treated with 2 Amol/L mismatch control
oligonucleotide or GRN163L for10 d and
the cells in each treatment flask were then
dividedinto two aliquots. Cells were then
cultured either in the presence of mismatch
or match (GRN163L) oligonucleotides
alone or with addition of ritonavir
(2 Amol/L; I) or doxorubicin (2 nmol/L; II).
Live cellnumber was determined at
different time points. B, in vivo efficacy of
GRN163L in a subcutaneous tumor model.
SCIDmice were inoculated s.c. in the
interscapular area with 3.0?106SEG-1
cells; following the appearance of tumors,
the mice were treatedi.p. with saline alone
or GRN163L 45 mg/kg/d.
Cancer Therapy: Preclinical
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subunit of telomerase (44) in Barrett’s adenocarcinoma. Our
data also show that telomeres are consistently shorter in BEAC
relative to normal esophageal epithelial cells. This is also
consistent with the observations of Finley et al. (56), indicating
shorter telomeres in BEAC relative to those in epithelial cells
from gastroesophageal reflux disease patients. Elevated telomer-
ase activity and shorter telomeres have also been reported in
multiple myeloma (40), B-cell chronic lymphocytic leukemia
(57), nonhematopoietic cancers such as hepatocellular carcino-
ma (58), breast cancer (59), and prostate cancer (60).
Although a variety of agents have been shown to inhibit
telomerase activity and block proliferation of cancer cells
(8, 9, 38, 42, 61–66), very few have been proven to be both
specific and suitable for in vivo delivery and utilization. The
telomerase inhibitor (GRN163L) used in this study is a thio-
phosphoramidate oligonucleotide complementary to the tem-
plate region of the RNA subunit of telomerase (hTERC). This
oligonucleotide has a lipid (C16) moiety attached to it, which
facilitates the uptake of this molecule in human cells without
any need of a transfection procedure or reagent. Using TAMRA-
labeled GRN163L, we have shown that this drug is efficiently
taken up by human adenocarcinoma cells without any
transfection within 24 hours of treatment. Uptake of drug
was associated with loss of telomerase activity and growth arrest
after a lag period of 3 to 4 weeks. Although for cancers such as
cervical cancer, the inhibition of telomerase induces apoptosis
within a few days without any requirement of telomere
shortening (67), the cell death in a majority of cancer cells
occurs after a lag period of a few weeks, which is probably
required for reduction of telomere length below critical limit
(8, 9, 38, 39, 42, 68). Because the treatment of adenocarcinoma
cells with GRN163L was associated with both the lag period
and reduction in telomere length, it seems that growth arrest
was probably induced by short telomeres.
Critically short telomeres are recognized as DNA damage and
therefore activate mechanisms leading to apoptotic cell death
or replicative senescence. Consistent with this, the inhibitors of
telomerase have been shown to induce apoptotic cell death (9,
38, 39, 69, 70) or both the apoptosis and senescence (8, 69) in
different cancer cell lines. In our study, the adenocarcinoma
cells treated with GRN163L stained positive for not only
Annexin V but also for h-galactosidase, indicating that growth
inhibition in these cells was associated with induction of both
the senescence and apoptosis. Induction of both the senescence
and apoptosis in adenocarcinoma cells following inhibition of
telomerase is consistent with our earlier observations (39).
However, we would caution that the data do not show that
both the senescence and apoptosis were induced simultaneous-
ly. Senescence may have preceded apoptosis following inhibi-
tion of telomerase, as observed by Herbert at al. (69), in
immortal breast epithelial cell line. This is quite consistent with
our data showing senescence in 64% and apoptosis in 85% of
treated SEG-1 cells.
We have also shown that growth inhibition following
exposure of adenocarcinoma cells to a telomerase inhibitor can
be significantly expedited by combination therapy with other
agents affecting telomerase or telomeres. The rationale of doing a
combination study was to first initiate the destruction of
telomericDNA in cancer cells by treating with a specific inhibitor
(GRN163L) and then expedite the degradation of vulnerable
telomeres by a very brief exposure to an agent, which may be less
specific, but could significantly expedite the destruction of
already eroding telomeres through different mechanisms. We
therefore chose a number of potential agents and evaluated their
effect on SEG-1 cells pretreated with GRN163L. Ritonavir and
doxorubicin were found to give a significant additive effect,
indicating the possibility of combination therapy with
GRN163L. The in vivo efficacy of GRN163L was shown in a
murine tumor model in which SCID mice were inoculated in the
interscapular area with SEG-1 adenocarcinoma cells and
following appearance of palpable tumors, mice were injected
GRN163L used has been shown by us previously to be nontoxic
and effective in two different tumor models of SCID mice (71).
Average tumor size in the mice treated with the drug was
>10-fold smaller than that in control mice (P = 0.03), without
evidence of toxicity, indicating a remarkable efficacy in vivo.
In summary, we have shown that telomerase activity assays
conducted in tissue extracts may produce inaccurate results and
propose that the activity assays should be conducted in the
lysates of cells purified by LCM. We have also shown that
telomerase activity is significantly elevated whereas telomere
length is significantly shorter in primary epithelial cells derived
from tissue specimens of BEAC relative to normal esophageal
epithelial cells. GRN163L, a lipid-conjugated oligonucleotide
thio-phosphoramidate targeting RNA component of telomer-
ase, is efficiently taken up by human adenocarcinoma cells
without any need of a transfection reagent/procedure, inhibits
telomerase activity, and induces telomere shortening and
growth inhibition by induction of both cellular senescence
and apoptosis. The growth inhibition following treatment with
GRN163L could also be expedited by a combination therapy
with other drugs. Finally, the efficacy of GRN163L has also
been shown in vivo. These data show that telomerase is a
potential target for BEAC and GRN163L is a potent and specific
telomerase inhibitor that, either alone or in combination with
agents such as ritonavir and doxorubicin, is suited for in vivo
utilization in human clinical trials.
Disclosure of Potential Conflicts of Interest
S. Gryaznovis employedby Geron Corporation andis co-inventorof GRN163L.
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