CLI NI CAL STUDI ES

Telomerase activity, telomere lengthand humantelomerase reverse

transcriptase expression in hepatocellular carcinoma is independent of
hepatitis virus status
Nitin Saini
1,
, Radhika Srinivasan
2
, Yogesh Chawla
1
, Sanjeev Sharma
1
, Anuradha Chakraborti
3
and
Arvind Rajwanshi
2
1 Department of Hepatology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
2 Department of Cytopathology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
3 Department of Experimental Medicine & Biotechnology, Postgraduate Institute of Medical Education & Research, Chandigarh, India
Keywords
hepatitis hepatocellular carcinoma
telomerase telomeres
Abbreviations
HBV, hepatitis B virus; HCC, hepatocellular
carcinoma; HCV, hepatitis C virus; hTERT,
human telomerase reverse transcriptase;
MTL, mean telomere length; NBNC-HCC,
non-B non-C hepatocellular carcinoma; TRAs,
telomere repeat arrays.
Correspondence
Prof. Yogesh Chawla, Department of
Hepatology, Postgraduate Institute of Medical
Education & Research, Chandigarh 160012,
India
Tel: 191 172 2756339
Fax: 191 172 2744401
e-mail: nitin3112k@yahoo.com
Received 13 December 2008
Accepted 12 June 2009
DOI:10.1111/j.1478-3231.2009.02082.x
Abstract
Background: Telomerase expression and the maintenance of a critical telomere
length (TL) in cancer initiation indicates that telomere shortening and
telomerase expression initiates cancer by induction of chromosomal instabil-
ity. Methods: Telomerase activity, TL and human telomerase reverse tran-
scriptase (hTERT) expression were investigated in 58 hepatocellular
carcinoma (HCC) and 17 chronic hepatitis patients by the telomerase repeat
amplication protocol, Southern blotting and reverse transcriptase-polymer-
ase chain reaction. Results: Telomerase was positive in 76% of HCC and
11.8% of chronic hepatitis patients (Po0.0001). The mean telomere length
(MTL) in HCC was signicantly shorter compared with chronic hepatitis
(P o0.0001). The MTL was not signicantly different in HCC patients with
and without cirrhosis (P=0.77). In hepatitis B virus, hepatitis C virus and
non-B non-C-related HCC groups, no differences were found in telomerase
activity and MTL (P =0.77). hTERT, a regulator of telomerase, was, however,
positive in 81% of HCCs. The correlation between telomerase activity and
hTERT mRNA expression was statistically signicant (P o0.0001). The MTL
in telomerase-positive HCC cases was signicantly shorter than the MTL in
telomerase-negative cases (Po0.0001). Conclusion: The majority of HCCs
exhibited telomerase activity that correlated well with hTERTexpression. MTL
in HCC was signicantly shorter than chronic hepatitis. It was also found that
shorter telomeres are present in telomerase-positive HCC cases. However, no
correlation was found between telomerase activity and TL with respect to the
viral status in HCC.
Hepatocellular carcinoma (HCC) is a common malig-
nancy worldwide and is the main cause of mortality in
patients with chronic liver disease, particularly in areas
having a high prevalence of chronic hepatitis B and C
viral infections (1, 2). Liver cirrhosis is a major risk factor
for the development of HCC. Abnormalities in the
known oncogenes and anti-oncogenes in HCC have not
been reported, except for some reports on the aberrant
p53 gene (3, 4). Thus, the mechanism of hepatocarcino-
genesis is still an enigma.
Telomeres are long repetitive stretches of DNA and
proteins, present at the ends of chromosomes, and are
considered to be important in protecting and stabilizing
the chromosomal ends. Telomeres consist of 520 kbp of
homogenous repeats of (TTAGGG)
n
(5) called telomere
repeat arrays (TRAs) (6). The telomere length (TL)
shortens at every cell division. Most normal somatic cells
lose approximately 50100 bp of telomeric repeat DNA
with each cycle of cell division (7, 8). When the TL reaches
a certain level, cell division stops and cell death occurs.
Therefore, the presence of telomeres of a certain length is
necessary for immortalization of cells. Differences in TLs
have been observed in tumour tissues of various cancers
when compared with the non-tumorous tissues (9).
As opposed to normal cells, immortalized cells in
culture and cancer cells in vivo exhibit short but sus-
tained TLs, which are maintained by the action of
telomerase, a reverse transcriptase with an RNA compo-
nent (10). Thus, telomerase is thought to be essential for
the acquisition of cellular immortality. It is a multimeric

Present address: Department of Cancer Biology, Kimmel Cancer Center,

233 South, BLSB Room No. 1008, Thomas Jefferson University,
Philadelphia, PA 19107, USA.
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Liver International ISSN 1478-3223
enzyme with three components, namely, human telo-
merase RNA component (hTERC), human telomerase-
associated protein 1 (hTEP1) and human telomerase
reverse transcriptase (hTERT) (1113). Of these, hTERT
has been proved to be a catalytic core protein component
of telomerase and is crucial for the regulation of telomer-
ase activity (13).
In HCC, telomerase and hTERT have been detected in
8091% of cases (14, 15). Telomerase has also been shown
to be present in more than 80% of macronodular poten-
tially precancerous hepatic lesions (16). The results in
cirrhotic tissue and chronic hepatitis are conicting (17).
There are no available studies comparing the status of
telomerase activity, TL and hTERTwith the viral status in
HCC and chronic hepatitis patients.
Material and methods
The study protocol conformed to the ethical guidelines
of the 1975 declaration of Helsinki as reected in a priori
approval by the institutions ethics committee, and all the
patients studied provided informed consent.
Liver samples
A total of 75 patients were studied, 58 with HCC (Group
I) and 17 chronic hepatitis without HCC (Group II).
There were 52 ne-needle aspiration biopsy (FNAB)
samples and six surgical samples in group I and 17 liver
biopsies in group II. Samples were immediately frozen in
liquid nitrogen and stored at 80 1C until use. The
diagnosis of HCC was established histologically. Asso-
ciated cirrhosis in HCC patients was diagnosed if the
liver surface was irregular on imaging (computed tomo-
graphy/magnetic resonance imaging) with a caudate lobe
enlargement. Thirty-two patients showed evidence of
associated cirrhosis, 20 had no cirrhosis, while in six,
evidence of cirrhosis could not be determined. Patients
with HCC were further grouped into those with solitary/
multiple lesions and small (o5 cm)/large (Z5 cm) HCC,
with and without portal vein involvement based on the
radiological ndings. All HCC cases were categorized
according to the Okuda classication (18).
There were 47 (81%) men and 11 (19%) women in
group I. Their ages ranged from 29 to 85 years
(56.71 11.84 years). In group II, 15 (80%) were men
and two (12%) were women. Their ages ranged from 11
to 58 years (44.24 12 years). In group I, the HBsAg was
positive in 34 (59%), antibody to the hepatitis C virus
(anti-HCV) in 12 (20.5%) patients and 12 (20.5%)
patients were negative for both viral markers. In group
II, 10 had HCV-related and seven had hepatitis B virus
(HBV)-related chronic hepatitis.
Measurement of telomerase activity
Telomerase activity was measured by a commercially
available telomerase polymerase chain reaction (PCR)
ELISA kit (Roche Diagnostics, Mannheim, Germany).
The kit was based on telomerase repeat amplication
protocol assay (10). Briey, liver specimens (FNAB,
biopsy and surgical samples) were homogenized in
200 ml of cold lysis buffer. The lysate was centrifuged at
16 000 g for 20 min at 4 1C. The supernatant was removed
and frozen in liquid nitrogen and stored at 80 1C. The
protein concentration was measured in each lysate by the
bicinchoninic acid protein assay kit (Bio Rad Labora-
tories, Hercules, CA, USA), and 6 mg of cellular protein
was used for the telomerase assay.
In each case, 46 ml of the lysate was incubated in a
50 ml reaction mixture containing 25 ml of reaction buffer
[20 mmol/L Tris-HCl, pH 8.3; 1.5 mmol/L MgCl
2
,
68 mmol/L KCl, 0.05% Tween 20, 1 mmol/L EGTA,
50 mmol/L deoxynucleotide triphosphate, TS primer,
(5
0
-AATCCGTCGAGCAGAGTT-3
0
), CX primer (5
0
-CC
CTTACCCTTACCCTTACCCTAA-3
0
), 2 U Taq DNA
polymerase and 0.5 mmol/L T4-gene-32 protein], 5 ml of
internal standard and the remaining PCR-grade water.
The mixture was incubated for 30 min at 25 1C and
then subjected to 31 PCR cycles at 94 1C 30 s, 50 1C 45 s
and 72 1C 90 s (nal cycle, 10 min at 72 1C). Products
were analysed on a 12.5% polyacrylamide non-denatur-
ing gel and silver stained. Finally, the gel was scanned and
photographed. If the characteristic six-nucleotide ladder
pattern was present in the gel, then it indicated positive
telomerase activity.
To examine the specicity of telomerase activity, heat-
inactivated (10 min at 100 1C) lysate in each case was
used to perform the assay and these heat-inactivated
samples were taken as negative controls.
Determination of telomere length
Telomere length was measured by a commercially avail-
able TL assay kit (Roche Diagnostics), based on the
Southern hybridization technique. Briey, genomic
DNA was extracted from the specimens, and quantied.
12 mg of DNA was digested for 2 h at 37 1C with 20 U
each of HinfI and RsaI. The sequence specicity of these
enzymes ensures that telomeric DNA and subtelomeric
DNA is not cut, while non-telomeric DNA is digested to
low-molecular-weight fragments. Digested DNAwas run
on a 0.8% agarose gel with a constant 5 V/cm power
supply for up to 10 cm gel length. Digoxigenin (DIG)-
labelled molecular weight markers were also run with
each lot of samples. After electrophoresis, DNA was
transferred overnight to a hybond
1
membrane (Amer-
sham Biosciences, Little Chalfont, UK). Transferred DNA
was xed by UV crosslinking (120 mJ, 2 min).
The blotted membranes containing the digested DNA
fragments were hybridized for 3 h at 45 1C in hybridiza-
tion buffer to a DIG-labelled probe specic for telomeric
repeats and incubated with a DIG-specic antibody
covalently coupled to alkaline phosphate. Finally, the
alkaline phosphatase on the antibody metabolized, yield-
ing a highly sensitive chemiluminescent substrate; this
produced a visible signal when exposed to an X-ray lm,
Liver International (2009)
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Saini et al. Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma
which indicated the location of the immobilized telo-
mere probe on the blot. Finally, the average/mean
telomere length (MTL) was determined by comparing
the mean location of the telomeric smear on the blot
relative to molecular weight standards on the same blot.
Expression of human telomerase reverse transcriptase
mRNA
Total RNA was extracted by the Qiagen kit (Qiagen
GmbH, Hilden, Germany) and checked by formaldehyde
agarose gel electrophoresis. cDNA was synthesized with
1 mg of total RNA. The hTERT gene was amplied by
using 3 ml of cDNA as a template with the sense primer
5
0
-CGGAAGAGTGTCTGGAGCAA-3
0
and the antisense
primer 5
0
-GGATGAAGCGGAGTCTGGA-3
0
(95 1C 5 min;
40 cycles, 95 1C 1 min, 52 1C 1 min, 72 1C 1 min 30 s; nal
extension, 72 1C 4 min). To ensure that the RNA was not
degraded a PCR assay for the b-actin gene was also
performed.
We conrmed that no contamination of the genomic
DNA existed by treating RNA samples with deoxyribo-
nuclease before reverse transcriptase-PCR.
Statistical analysis
All the experiments were performed in duplicate and the
reproducibility was conrmed. Telomerase activity and
the hTERT mRNA expression were compared between
the different groups by using the w
2
-test, and TLs were
compared with the MannWhitney U-test. Fishers exact
test was used for the correlation between hTERT and
telomerase activity. One-way ANOVA (a non-parametric
alternative KruskalWallis test) was used to test the
association between the MTL and Okuda staging. A P-
value of 0.05 or less was considered significant.
Results
Demographical data
Demographical data of the patients in the two groups are
shown in Table 1.
Telomerase activity in hepatocellular carcinoma and
chronic hepatitis
Telomerase activity was present in 76% (44/58) of the
HCC samples (supporting information Table S1, Fig. 1A
and B). In contrast, telomerase activity was found in only
two (11.8%) of 17 chronic hepatitis cases (Po0.0001)
(supporting information Table S2, Fig. 2).
Of the 27 HCC patients with a solitary lesion, 20
(74.15%) were positive for telomerase compared with 23
of the 29 HCC patients (79.3%), who had multiple
lesions. This difference was not statistically signicant
(P=0.44). Eleven of the 12 (91.67%) patients with a
tumour size of o5 cm were positive for telomerase
activity, while 32 of the 44 (72.72%) with a tumour size
of Z5 cm were found to be positive for telomerase
activity, the difference not being signicant (P=0.36).
Okuda classication was performed in 56 out of 58
patients and eight (14.3%) patients were in stage I, 42
(75%) in stage II and six (10.7%) in stage III. However,
there was no relationship of telomerase activity with the
Okuda stage of the HCC (P=0.87) (Table 2).
Portal vein thrombosis (PVT) was present in 34 (73%)
out of 47 HCC patients in whom a proper assessment of
the PV could be performed. We did not nd any
signicant correlation of the presence of PVT with
telomerase activity in HCC (Table 1).
In 34 HBsAg-positive HCCs, telomerase activity was
present in 24 (70.6%) while in HCV-related and non-B
non-C (NBNC) HCC cases, the positivity rate was 75%
(9/12) and 91.7% (11/12) respectively (supporting in-
formation Table S1). Telomerase activity was positive in
72% of (23/32) HCC patients who had associated
cirrhosis and in 80% (16/20) of those HCCs without
underlying cirrhosis (Table 2). Thus, the presence of viral
markers and cirrhosis in HCC patients did not have any
correlation with telomerase activity.
Expression of human telomerase reverse transcriptase
mRNA
Human telomerase reverse transcriptase mRNA was
detected in 81% of HCCs (supporting information Table
S1, Fig. 3) and in 11.8% of chronic hepatitis patients
Table 1. Demographical and clinical prole of hepatocellular carci-
noma and chronic hepatitis cases
HCC (%)
Chronic hepatitis
(%)
Mean age (years) 56.7111.84 44.2412
(Range, 2985) (Range, 1150)
Male:Female 4.27:1 7.5:1
Viral marker
Hepatitis B 34 (58.6) 7 (41.2)
Hepatitis C 12 (20.7) 10 (58.8)
NBNC 12 (20.7)
Cirrhosis (52)

Present 32 (61.5)
Not present 20 (38.5)
Tumour number (56)

Single 27 (48.2)
Multiple (Z2) 29 (51.8)
Tumour size (56)

o5cm 12 (21.4)
Z5 cm 44 (78.6)
Okuda stage (56)

I 8 (14.3)
II 42 (75)
III 6 (10.7)
Portal vein thrombosis (47)

Present 34 (73)
Absent 13 (27)

Number of cases in whom the investigation was performed.

HCC, hepatocellular carcinoma; NBNC, non-B non-C.
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Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma Saini et al.
(supporting information Table S2, Fig. 4); the difference
was statistically signicant (Po0.0001). Three patients
of HCC who were negative for telomerase activity were
positive for hTERT mRNA expression. Overall, the
expression of hTERT mRNA was signicantly related to
telomerase activity (P o0.0001).
Fig. 1. (A) Telomerase repeat amplication protocol assay in hepatocellular carcinoma (HCC) cases. Lane P, positive control; lanes 16, HCC
samples; lane T, test samples; lane N, heat-inactivated negative control of that sample. The arrow shows the amplication of the 216bp
internal standard. Telomerase activity is evident by the 6 bp-specic ladder pattern in all the cases. (B) Representative HCC cases for telomerase
activity determination. Lanes 15, HCC samples; lane T, test samples; lane N, heat-inactivated negative control of that sample. Note: In case no.
3, there is no telomerase activity as evident by the complete absence of the characteristic ladder pattern.
Fig. 2. Positive telomerase repeat amplication protocol assay in
two cases of chronic hepatitis. Lane P, positive control; lanes 1 and 2,
chronic hepatitis samples; lane T, test sample; lane N, heat-
inactivated negative control of that sample. The arrow shows the
amplication of the 216bp internal standard.
Table 2. Comparison of telomerase activity in different subsets of
hepatocellular carcinomas and in chronic hepatitis cases
Group Telomerase positivity (%) P value
HCC cases (n =58) 44 (76) o0.0001
Chronic hepatitis (n =17) 2 (11.8)
HCCs

Single lesion (n =27) 20 (74) NS (0.44)

Multiple lesions (n =29) 23 (79.3)
HCCs

o5 cm (n =12) 11 (91.67) NS (0.36)

Z5 cm (n =44) 32 (72.72)
Cirrhosis

Present (n =32) 23 (72) NS (0.32)

Absent (n =20) 16 (80)
Okuda stage

I (n =8) 6 (75) NS (0.87)

II (n =42) 34 (81)
III (n=6) 4 (66.7)

All had a signicantly higher telomerase positivity rate when compared

with the chronic hepatitis group (P o0.0001); Telomerase activity was
signicantly correlated to the hTERT transcript expression (P o0.0001).
HCC, hepatocellular carcinoma; hTERT, human telomerase reverse tran-
scriptase; NS, non-signicant.
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Saini et al. Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma
Telomere length in hepatocellular carcinoma and chronic
hepatitis
The MTL was estimated in 54 HCC patients. In four
samples, estimation was not possible for technical rea-
sons. In HCC, MTLwas 4.81 2.31 kb (range, 2.014.50)
compared with 7.04 1.10 kb (5.5010.40) in patients
with chronic hepatitis (Table 3, Fig. 5). Thus, the TL in
HCC was signicantly shorter than that in chronic
hepatitis (P o0.0001). The TLs in HCC were also more
widely distributed than those in chronic hepatitis cases
(Fig. 6).
Of the 52 HCC patients, MTL in 32 (61.5%) patients
with cirrhosis had a shorter TL [4.69 2.09 kb (range,
2.0012.20)] compared with 20 patients with a non-
cirrhotic background [MTL, 5.04 2.74 kb (range,
2.0014.50)]. Even though the difference between the
above two groups was not signicant (Table 3), a trend
towards shorter TL was seen in cirrhotic HCC cases
(P=0.77) (Fig. 7). There was also no signicant differ-
ence in the MTL with respect to the Okuda staging
(P=0.60). There was no difference in the MTL in HCC
patients with PVT and those without (4.20 2.12 vs
4.92 1.82). The MTL in HBV-related HCC was
4.95 2.14 vs 4.92 3.75 kb in HCV related and
Fig. 3. Human telomerase reverse transcriptase (hTERT) mRNA
expression in hepatocellular carcinoma cases. M, molecular weight
markers; lanes 17, hepatocellular carcinoma (HCC) samples. The
upper panel shows the hTERT transcript whereas the lower panel
represents the corresponding b-actin transcript. The hTERT mRNA
was absent in one of the HCC cases (lane 7).
Fig. 4. Human telomerase reverse transcriptase (hTERT) mRNA
expression in chronic hepatitis cases. M, molecular weight markers;
lanes 16, chronic hepatitis samples. The upper panel shows the
hTERT transcript, whereas the lower panel represents the
corresponding b-actin transcript. The hTERT mRNA was present in
two of the chronic hepatitis cases (lanes 1 and 2).
Table 3. Mean telomere length in different subset of hepatocellular
carcinomas and in chronic hepatitis cases, classied according to
their viral aetiologies, size of tumour, number of lesions and
necroinammatory and brosis scores
Groups
Telomere length
MeanSD (kb) P value
HCC 4.812.31 o0.0001
Chronic hepatitis
a
7.041.10
HBV HCC
b
4.952.14 b vs a, o0.0001
HCV HCC
c
4.923.75 c vs a, 0.038
NBNC-HCC
d
4.340.96 d vs a, o0.0001
HCC
Single lesion 5.02.58 0.670 (NS)
Multiple lesions 4.652.16
HCC
o5cm 4.051.72 0.335 (NS)
Z5 cm 4.972.53
HCC
Cirrhosis present 4.692.09 0.77 (NS)
Absent 5.042.74
Okuda stage (HCCs)
I 4.571.70
II 4.652.18 0.60 (NS)
III 6.203.62
Portal vein thrombosis
Present 4.202.12 0.36 (NS)
Absent 4.921.82
Chronic hepatitis
NI 3 7.161.45 0.650 (NS)
43 6.900.60
Chronic hepatitis
F 2 6.941.59 0.744 (NS)
42 7.120.49
b vs c =NS; c vs d=NS; b vs d=NS.
F, brosis; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV,
hepatitis C virus; NI, necroinammatory; NBNC, non-B non-C; NS, non-
signicant.
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Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma Saini et al.
4.34 0.96 kb in NBNC-HCCs, which were not signi-
cantly different from each other (P =0.77).
Correlation between telomerase activity and telomere
length in hepatocellular carcinoma
In 54 patients with HCC, telomerase activity as well as TL
were determined. These included 14 cases negative for
telomerase activity and 40 positive for telomerase activ-
ity. The MTL in telomerase-negative HCC was
6.53 2.15 kb (range, 2.7012.20) whereas in telomer-
ase-positive HCC, it was 4.20 2.06 kb (range,
2.0014.50). Interestingly, this difference was statistically
signicant (P o0.0001), implying shorter telomeres in
telomerase-positive tumours (Fig. 8).
Discussion
Hepatocellular carcinoma is one of the most prevalent
cancers in the world, but the precise mechanism(s) of
Fig. 5. Telomere length estimation by Southern blotting in
hepatocellular carcinoma (upper panel) and chronic hepatitis (lower
panel) cases. The upper panel shows the telomere lengths (TLs) in
hepatocellular carcinoma (HCC) cases and the lower panel shows
the TL in chronic hepatitis cases; MWM, digoxigenin-labelled
molecular weight markers.
Fig. 6. Telomere length distribution in chronic hepatitis (CH) and
hepatocellular carcinoma (HCC) cases.
Fig. 7. Telomere length comparison in cases of chronic hepatitis
(CH) and hepatocellular carcinoma (HCC) with or without cirrhosis.
NS, non-signicant.
Fig. 8. Comparative analysis of telomerase activity and telomere
length in hepatocellular carcinoma cases with and without cirrhosis.
( ), hepatocellular carcinoma (HCC) cases negative for telomerase
activity; (1), HCC cases positive for telomerase activity; NS, non-
signicant.
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Saini et al. Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma
liver carcinogenesis is still not clearly understood (19). In
the present study, we evaluated the telomere status with
respect to the underlying hepatitis virus infection in the
cases of HCC and chronic viral hepatitis without HCC.
The overall telomerase positivity rate was 76% in HCCs
and 11.8% in chronic hepatitis. In most previous reports
on various malignant tissues, the telomerase activity was
typically found to be positive in almost 6585% of cases;
however, it is not necessarily positive in all cases (2022).
In fact, one report has clearly shown that several tumour
cell lines retain their TL without telomerase activity, and
there possibly is some other unidentied mechanism that
restores the TL (23). Thus, it seems that a small number
of HCCs actually do not possess telomerase activity.
However, the positive rate for telomerase was concordant
with the previous reports on patients with HCC by other
groups, which typically lies between 80 and 85% (14, 17).
The above ndings support the fact that telomerase is
activated in cancer. Telomerase activity was also detected
in two (11.8%) of 17 chronic hepatitis cases. Although we
do not know the exact reason for the detectability of
telomerase activity in chronic hepatitis, it may be because
of the presence of increased brosis and necrosis in these
as both of these cases had high histological activity index
scores (supporting information Table S2).
In the present study, we compared the telomerase
activity between HCC patients classied according to
their viral aetiologies. However, the results were not
statistically signicant but interestingly HBsAg-positive
HCC patients and HCV-related HCCs were less likely to
express telomerase (70.6 and 75%) than NBNC-HCC
patients (91.7%). This could be explained as most of
our NBNC-HCC patients were also suspected to have
diabetes as depicted from their high fasting blood glucose
levels (data not shown) and this hyperglycaemic situa-
tion can induce the generation of free radicals, which
may ultimately inuence the telomerase activity by
increasing oxidative stress (2426). These ndings were
exciting and have not been reported earlier, particularly
in view of enhanced telomerase positivity in NBNC-HCC
cases. However, a recent study showed that high telomer-
ase activity is a poor prognostic marker in HCC, but the
main drawback in this study is that it only included
HBV-related HCC cases (27). Nonetheless, these results
are exciting and further studies with a large number of
HCC cases, especially NBNC-related HCCs, need to be
carried out in this regard.
The expression of hTERT is rate limiting for telomer-
ase activity (28). In our study, hTERTmRNA expression
was found in 81% of HCCs and in 11.8% of chronic
hepatitis cases. The correlation between telomerase
activity and hTERTexpression was statistically signicant
(Po0.0001). In a previous study, expression of telomer-
ase-associated protein 1 and telomerase reverse tran-
scriptase was positively correlated in 23 HCC patients
(29).
In this study, the MTL of HCC tissues was signicantly
shorter than that of chronic hepatitis tissues. The main
function of telomeres is to act as protective caps at
the ends of chromosomes, thus preventing end-to-end
fusion of chromosomes and further chromosomal in-
stability. Telomere shortening promotes chromosomal
instability during ageing and chronic diseases. It occurs
during the course of chronic liver disease and is one of
the causes of hepatocarcinogenesis (14, 30). A range of
studies in different human cancers demonstrate shor-
tened telomeres in tumorous tissues (31). However,
carcinogenesis is believed to be a multistage process
driven by genetic damage and epigenetic changes. Thus,
a trend towards a shorter TL in cirrhotic HCC cases as
compared with non-cirrhotic HCC cases, which was seen
in this study, can be explained by the fact that these
patients have accumulated greater number of genetic
alterations during the process of cell cycles and/or
progression of the disease. Moreover, the risk of HCC
increases sharply at the cirrhosis stage that is character-
ized by hepatocyte telomere shortening, although this
would have been more clear if we had included a pure
cirrhotic group without any evidence of HCC in our
analysis. But, this was practically not possible as cirrhotic
patients were not amenable to any invasive procedures.
Our data support the telomere hypothesis that chronic
liver injury induces continual waves of liver destruction
and regeneration, resulting in critical telomere short-
ening, which in turn culminates in hepatocyte-replicative
senescence and ultimately in liver cirrhosis (32). In
humans, few studies have described the shortening of
telomeres in cirrhosis induced by viral hepatitis (33, 34).
A recent study by Zhang et al. (35) also found that TL in
HCC tissues was not correlated with other clinical
parameters, such as age, sex and HBV infection status,
but the same study only included HBV-associated HCC
and the sample number was also less. To our knowledge,
our paper is the rst to include an NBNC-HCCs group
along with HBV- and HCV-related groups, and exam-
ined whether or not the TL and telomerase activity is
different according to the aetiology of HCC, none of
which appear to differ between the different aetiologies
of HCC. We also did not nd any correlation of TL with
the Okuda staging of HCC. As such, the length of a
telomere in a cell type is partially determined genetically,
but several environmental factors also have effects on TL
including cumulative oxidative and inammatory and
psychological stress (25).
More specically, the mechanism of HBV-related HCC
can be mainly attributed to the integration of the viral
genome into the hepatocyte chromosomal DNA, thereby
leading to rearrangement and instability of the host DNA.
Recent investigations have shown that the telomerase
gene is targeted for integration in different HBV-related
HCCs, suggesting common pathways in HBV-related
carcinogenesis (36). While the involvement of HCV in
hepatocarcinogenesis can be explained by involvement of
the HCV core protein, leading to the overproduction of
oxidative stress, which yields genetic aberrations, modu-
lation of cellular gene expressions and altered intracellular
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Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma Saini et al.
signal transductions, the combination of these alterations
would be hypothesized to promote the development of
HCC in HCV infection (37). However, the cause of
NBNC-HCC can be mainly attributed to the persistent
existence of diabetes and/or non-alcoholic steatohepatitis
as an underlying ailment. In our chronic hepatitis cases,
the MTL was found to be 7.04 1.10 kb, which is slightly
shorter than that reported previously (7.30 1.40 and
8.90 1.74 kb respectively) (14, 33). This may be because
of the differences in age and the clinical stage of chronic
hepatitis patients.
A signicant correlation of shorter telomeres with
positive telomerase activity was found in our study (Fig.
8). This observation was interesting and has not been
reported earlier. Usually, when the length of a telomere is
long enough for proliferation, telomerase activity sub-
sides (38) but some mammalian cells, also without any
telomerase activity, are able to maintain the length of
their telomeres for many population doublings (23), thus
indicating the existence of one or more non-telomerase
mechanism(s) for telomere maintenance that have been
termed alternative lengthening of telomeres (ALT) (23,
39). To date, clear evidence for ALTactivity has only been
found in some immortalized human cell lines and in
telomerase-null mouse cell lines (40, 41). It seems likely
that understanding this form of telomere maintenance in
HCC may have important implications for the diagnosis
and treatment of HCC. Further studies are required to
conrm this form of telomere maintenance in HCC.
Acknowledgements
The authors acknowledge the nancial support provided
by the Indian Council of Medical Research (ICMR) for
this study.
Disclosures: None.
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Supporting information
Additional supporting information may be found in the
online version of this article:
Table S1. Telomerase activity, telomere length and
hTERTmRNA expression in 58 hepatocellular carcinoma
cases.
Table S2. Telomerase activity, telomere length and
hTERTmRNA expression in 17 chronic hepatitis cases.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supporting materials
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material) should be directed to the corresponding author
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Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma Saini et al.