telomerase activity, telomere length and human telomerase reverse

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C L I N I C A L S T U D I E S

Telomeraseactivity, telomere lengthandhuman telomerase reversetranscriptaseexpression in hepatocellular carcinoma is independentofhepatitisvirusstatus

Nitin Saini1,, Radhika Srinivasan2, Yogesh Chawla1, Sanjeev Sharma1, Anuradha Chakraborti3 andArvind Rajwanshi2

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:[email protected]

Received 13 December 2008Accepted 12 June 2009

DOI:10.1111/j.1478-3231.2009.02082.x

AbstractBackground: Telomerase expression and the maintenance of a critical telomerelength (TL) in cancer initiation indicates that telomere shortening andtelomerase 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 repeatamplification protocol, Southern blotting and reverse transcriptase-polymer-ase chain reaction. Results: Telomerase was positive in 76% of HCC and11.8% of chronic hepatitis patients (Po 0.0001). The mean telomere length(MTL) in HCC was significantly shorter compared with chronic hepatitis(Po 0.0001). The MTL was not significantly different in HCC patients withand without cirrhosis (P= 0.77). In hepatitis B virus, hepatitis C virus andnon-B non-C-related HCC groups, no differences were found in telomeraseactivity and MTL (P= 0.77). hTERT, a regulator of telomerase, was, however,positive in 81% of HCCs. The correlation between telomerase activity andhTERT mRNA expression was statistically significant (Po 0.0001). The MTLin telomerase-positive HCC cases was significantly shorter than the MTL intelomerase-negative cases (Po 0.0001). Conclusion: The majority of HCCsexhibited telomerase activity that correlated well with hTERTexpression. MTL

in HCC was significantly shorter than chronic hepatitis. It was also found thatshorter telomeres are present in telomerase-positive HCC cases. However, nocorrelation was found between telomerase activity and TL with respect to theviral status in HCC.

Hepatocellular carcinoma (HCC) is a common malig-nancy worldwide and is the main cause of mortality inpatients with chronic liver disease, particularly in areashaving a high prevalence of chronic hepatitis B and Cviral infections (1, 2). Liver cirrhosis is a major risk factorfor the development of HCC. Abnormalities in theknown oncogenes and anti-oncogenes in HCC have not

been reported, except for some reports on the aberrantp53 gene (3, 4). Thus, the mechanism of hepatocarcino-genesis is still an enigma.

Telomeres are long repetitive stretches of DNA andproteins, present at the ends of chromosomes, and areconsidered to be important in protecting and stabilizing

the chromosomal ends. Telomeres consist of 520 kbp ofhomogenous repeats of (TTAGGG)

n(5) called telomere

repeat arrays (TRAs) (6). The telomere length (TL)shortens at every cell division. Most normal somatic cellslose approximately 50100bp of telomeric repeat DNAwith each cycle of cell division (7, 8). When the TL reachesa certain level, cell division stops and cell death occurs.

Therefore, the presence of telomeres of a certain length isnecessary for immortalization of cells. Differences in TLshave been observed in tumour tissues of various cancerswhen compared with the non-tumorous tissues (9).

As opposed to normal cells, immortalized cells inculture and cancer cells in vivo exhibit short but sus-tained TLs, which are maintained by the action oftelomerase, a reverse transcriptase with an RNA compo-nent (10). Thus, telomerase is thought to be essential forthe 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
mailto:[email protected]:[email protected]


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enzyme with three components, namely, human telo-merase RNA component (hTERC), human telomerase-associated protein 1 (hTEP1) and human telomerasereverse transcriptase (hTERT) (1113). Of these, hTERThas been proved to be a catalytic core protein componentof telomerase and is crucial for the regulation of telomer-

ase activity (13).In HCC, telomerase and hTERT have been detected in8091% of cases (14, 15). Telomerase has also been shownto be present in more than 80% of macronodular poten-tially precancerous hepatic lesions (16). The results incirrhotic tissue and chronic hepatitis are conflicting (17).

There are no available studies comparing the status oftelomerase activity, TL and hTERTwith the viral status inHCC and chronic hepatitis patients.

Material and methods

The study protocol conformed to the ethical guidelinesof the 1975 declaration of Helsinki as reflected in a prioriapproval 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 (GroupI) and 17 chronic hepatitis without HCC (Group II).There were 52 fine-needle aspiration biopsy (FNAB)samples and six surgical samples in group I and 17 liverbiopsies in group II. Samples were immediately frozen inliquid nitrogen and stored at 80 1C until use. Thediagnosis of HCC was established histologically. Asso-ciated cirrhosis in HCC patients was diagnosed if theliver surface was irregular on imaging (computed tomo-graphy/magnetic resonance imaging) with a caudate lobe

enlargement. Thirty-two patients showed evidence ofassociated cirrhosis, 20 had no cirrhosis, while in six,evidence of cirrhosis could not be determined. Patientswith HCC were further grouped into those with solitary/multiple lesions and small (o 5 cm)/large (Z5 cm) HCC,with and without portal vein involvement based on theradiological findings. All HCC cases were categorizedaccording to the Okuda classification (18).

There were 47 (81%) men and 11 (19%) women ingroup I. Their ages ranged from 29 to 85 years(56.71 11.84 years). In group II, 15 (80%) were menand two (12%) were women. Their ages ranged from 11to 58 years (44.24 12 years). In group I, the HBsAg waspositive 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 groupII, 10 had HCV-related and seven had hepatitis B virus(HBV)-related chronic hepatitis.

Measurement of telomerase activity

Telomerase activity was measured by a commerciallyavailable telomerase polymerase chain reaction (PCR)ELISA kit (Roche Diagnostics, Mannheim, Germany).

The kit was based on telomerase repeat amplificationprotocol assay (10). Briefly, liver specimens (FNAB,biopsy and surgical samples) were homogenized in200ml of cold lysis buffer. The lysate was centrifuged at16 000gfor 20min at 4 1C. The supernatant was removedand frozen in liquid nitrogen and stored at 80 1C. The

protein concentration was measured in each lysate by thebicinchoninic acid protein assay kit (Bio Rad Labora-tories, Hercules, CA, USA), and 6 mg of cellular proteinwas used for the telomerase assay.

In each case, 46 ml of the lysate was incubated in a50ml reaction mixture containing 25 ml of reaction buffer[20 mmol/L Tris-HCl, pH 8.3; 1.5 mmol/L MgCl2,68 mmol/L KCl, 0.05% Tween 20, 1 mmol/L EGTA,50mmol/L deoxynucleotide triphosphate, TS primer,(50-AATCCGTCGAGCAGAGTT-30), CX primer (50-CCCTTACCCTTACCCTTACCCTAA-3 0), 2 U Taq DNApolymerase and 0.5mmol/L T4-gene-32 protein], 5 ml ofinternal 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 941C 30 s, 50

1C 45 sand 72 1C 90s (final 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 andphotographed. If the characteristic six-nucleotide ladderpattern was present in the gel, then it indicated positivetelomerase activity.

To examine the specificity of telomerase activity, heat-inactivated (10min at 1001C) lysate in each case wasused to perform the assay and these heat-inactivatedsamples 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 theSouthern hybridization technique. Briefly, genomicDNA was extracted from the specimens, and quantified.12mg of DNA was digested for 2 h at 37 1C with 20Ueach ofHinfI andRsaI. The sequence specificity of theseenzymes ensures that telomeric DNA and subtelomericDNA is not cut, while non-telomeric DNA is digested tolow-molecular-weight fragments. Digested DNA was runon a 0.8% agarose gel with a constant 5 V/cm powersupply for up to 10 cm gel length. Digoxigenin (DIG)-labelled molecular weight markers were also run witheach lot of samples. After electrophoresis, DNA wastransferred overnight to a hybond1 membrane (Amer-

sham Biosciences, Little Chalfont, UK). Transferred DNAwas fixed 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 specific for telomericrepeats and incubated with a DIG-specific antibodycovalently coupled to alkaline phosphate. Finally, thealkaline phosphatase on the antibody metabolized, yield-ing a highly sensitive chemiluminescent substrate; thisproduced a visible signal when exposed to an X-ray film,

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Sainiet al. Telomerase activity, telomere length and hTERT expression in hepatocellular carcinoma


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which indicated the location of the immobilized telo-mere probe on the blot. Finally, the average/meantelomere length (MTL) was determined by comparingthe mean location of the telomeric smear on the blotrelative to molecular weight standards on the same blot.

Expression of human telomerase reverse transcriptasemRNA

Total RNA was extracted by the Qiagen kit (QiagenGmbH, Hilden, Germany) and checked by formaldehydeagarose gel electrophoresis. cDNA was synthesized with1mg of total RNA. The hTERT gene was amplified byusing 3 ml of cDNA as a template with the sense primer50-CGGAAGAGTGTCTGGAGCAA-30 and the antisenseprimer 50-GGATGAAGCGGAGTCTGGA-3 0 (951C 5 min;40 cycles, 95 1C 1 min, 521C 1 min, 721C 1 min 30 s; finalextension, 72 1C 4 min). To ensure that the RNA was notdegraded a PCR assay for the b-actin gene was alsoperformed.

We confirmed 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 thereproducibility was confirmed. Telomerase activity andthe hTERT mRNA expression were compared betweenthe different groups by using the w2-test, and TLs werecompared with the MannWhitneyU-test. Fishers exacttest was used for the correlation between hTERT andtelomerase activity. One-way ANOVA (a non-parametricalternative KruskalWallis test) was used to test theassociation 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 areshown in Table 1.

Telomerase activity in hepatocellular carcinoma andchronic hepatitis

Telomerase activity was present in 76% (44/58) of theHCC samples (supporting information Table S1, Fig. 1Aand B). In contrast, telomerase activity was found in only

two (11.8%) of 17 chronic hepatitis cases (Po

0.0001)(supporting information Table S2, Fig. 2).Of the 27 HCC patients with a solitary lesion, 20

(74.15%) were positive for telomerase compared with 23of the 29 HCC patients (79.3%), who had multiplelesions. This difference was not statistically significant(P= 0.44). Eleven of the 12 (91.67%) patients with atumour size of o 5 cm were positive for telomeraseactivity, while 32 of the 44 (72.72%) with a tumour sizeof Z5 cm were found to be positive for telomerase

activity, the difference not being significant (P= 0.36).Okuda classification was performed in 56 out of 58patients 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 theOkuda 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 ofthe PV could be performed. We did not find anysignificant correlation of the presence of PVT withtelomerase activity in HCC (Table 1).

In 34 HBsAg-positive HCCs, telomerase activity waspresent in 24 (70.6%) while in HCV-related and non-Bnon-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 in72% of (23/32) HCC patients who had associatedcirrhosis and in 80% (16/20) of those HCCs without

underlying cirrhosis (Table 2). Thus, the presence of viralmarkers and cirrhosis in HCC patients did not have anycorrelation with telomerase activity.

Expression of human telomerase reverse transcriptasemRNA

Human telomerase reverse transcriptase mRNA wasdetected in 81% of HCCs (supporting information TableS1, Fig. 3) and in 11.8% of chronic hepatitis patients

Table 1. Demographical and clinical profile 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 7.5:1Viral 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)

o5 cm 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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(supporting information Table S2, Fig. 4); the differencewas statistically significant (Po 0.0001). Three patientsof HCC who were negative for telomerase activity werepositive for hTERT mRNA expression. Overall, theexpression of hTERT mRNA was significantly related totelomerase activity (Po 0.0001).

Fig. 1. (A) Telomerase repeat amplification 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 amplification of the 216 bp

internal standard. Telomerase activity is evident by the 6 bp-specific ladder pattern in all the cases. (B) Representative HCC cases for telomerase

activity determination. Lanes 15,HCC samples; laneT, test samples; laneN, heat-inactivatednegative control of that sample. Note: In caseno.

3, there is no telomerase activity as evident by the complete absence of the characteristic ladder pattern.

Fig. 2. Positive telomerase repeat amplification protocol assay in

two casesof chronic hepatitis. Lane P, positive control; lanes 1 and2,

chronic hepatitis samples; lane T, test sample; lane N, heat-

inactivated negative control of that sample. The arrow shows the

amplification of the 216 bp internal standard.

Table 2. Comparison of telomerase activity in different subsets ofhepatocellular carcinomas and in chronic hepatitis cases

Group Telomerase positivity (%) Pvalue

HCC cases (n = 58) 44 (76) o0.0001

Chronic hepatitis (n = 1 7) 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 significantly higher telomerase positivity rate when compared

with the chronic hepatitis group (Po0.0001); Telomerase activity was

significantly correlated to the hTERT transcript expression (Po0.0001).HCC, hepatocellular carcinoma; hTERT, human telomerase reverse tran-

scriptase; NS, non-significant.




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Telomere length in hepatocellular carcinoma and chronichepatitis

The MTL was estimated in 54 HCC patients. In foursamples, estimation was not possible for technical rea-

sons. In HCC, MTL was 4.81 2.31 kb (range, 2.014.50)compared with 7.04 1.10kb (5.5010.40) in patientswith chronic hepatitis (Table 3, Fig. 5). Thus, the TL inHCC was significantly shorter than that in chronichepatitis (Po 0.0001). The TLs in HCC were also morewidely distributed than those in chronic hepatitis cases(Fig. 6).

Of the 52 HCC patients, MTL in 32 (61.5%) patientswith 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 2.74kb (range,2.0014.50)]. Even though the difference between theabove two groups was not significant (Table 3), a trendtowards shorter TL was seen in cirrhotic HCC cases(P= 0.77) (Fig. 7). There was also no significant differ-ence in the MTL with respect to the Okuda staging(P= 0.60). There was no difference in the MTL in HCCpatients with PVT and those without (4.20 2.12 vs4.92 1.82). The MTL in HBV-related HCC was4.95 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 weightmarkers; lanes 17, hepatocellular carcinoma (HCC) samples. The

upper panel shows the hTERT transcript whereas the lower panel

represents the correspondingb-actin transcript. The hTERT mRNA

was absent in one of the HCC cases (lane 7).

Fig. 4. Human telomerase reverse transcriptase (hTERT) mRNAexpression in chronic hepatitis cases. M, molecular weight markers;

lanes 16, chronic hepatitis samples. The upper panel shows thehTERT 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, classified according to

their viral aetiologies, size of tumour, number of lesions and

necroinflammatory and fibrosis scores

Groups

Telomere length

Mean SD (kb) Pvalue

HCC 4.812.31 o0.0001Chronic hepatitisa 7.041.10

HBV HCCb 4.952.14 bvsa, o0.0001HCV HCCc 4.923.75 cvsa, 0.038

NBNC-HCCd 4.340.96 dvsa, o0.0001HCC

Single lesion 5.02.58 0.670 (NS)

Multiple lesions 4.652.16

HCC

o5 cm 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

bvsc =NS;cvsd =NS;bvsd =NS.

F, fibrosis; HBV, hepatitis B virus; HCC, hepatocellular carcinoma; HCV,

hepatitis C virus; NI, necroinflammatory; NBNC, non-B non-C; NS, non-

significant.




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4.34 0.96kb in NBNC-HCCs, which were not signifi-cantly different from each other (P= 0.77).

Correlation between telomerase activity and telomerelength in hepatocellular carcinoma

In 54 patients with HCC, telomerase activity as well as TLwere determined. These included 14 cases negative fortelomerase activity and 40 positive for telomerase activ-ity. The MTL in telomerase-negative HCC was6.53 2.15kb (range, 2.7012.20) whereas in telomer-ase-positive HCC, it was 4.20 2.06kb (range,2.0014.50). Interestingly, this difference was statisticallysignificant (Po 0.0001), implying shorter telomeres intelomerase-positive tumours (Fig. 8).

Discussion

Hepatocellular carcinoma is one of the most prevalentcancers in the world, but the precise mechanism(s) of

Fig. 5. Telomere length estimation by Southern blotting inhepatocellular 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) andhepatocellular carcinoma (HCC) cases.

Fig. 7. Telomere length comparison in cases of chronic hepatitis

(CH) and hepatocellular carcinoma (HCC) with or without cirrhosis.

NS, non-significant.

Fig. 8. Comparative analysis of telomerase activity and telomerelength 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-

significant.




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liver carcinogenesis is still not clearly understood (19). Inthe present study, we evaluated the telomere status withrespect to the underlying hepatitis virus infection in thecases of HCC and chronic viral hepatitis without HCC.The overall telomerase positivity rate was 76% in HCCsand 11.8% in chronic hepatitis. In most previous reports

on various malignant tissues, the telomerase activity wastypically 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 tumourcell lines retain their TL without telomerase activity, andthere possibly is some other unidentified mechanism thatrestores the TL (23). Thus, it seems that a small numberof HCCs actually do not possess telomerase activity.However, the positive rate for telomerase was concordantwith the previous reports on patients with HCC by othergroups, which typically lies between 80 and 85% (14, 17).The above findings support the fact that telomerase isactivated in cancer. Telomerase activity was also detectedin two (11.8%) of 17 chronic hepatitis cases. Although we

do not know the exact reason for the detectability oftelomerase activity in chronic hepatitis, it may be becauseof the presence of increased fibrosis and necrosis in theseas both of these cases had high histological activity indexscores (supporting information Table S2).

In the present study, we compared the telomeraseactivity between HCC patients classified according totheir viral aetiologies. However, the results were notstatistically significant but interestingly HBsAg-positiveHCC patients and HCV-related HCCs were less likely toexpress telomerase (70.6 and 75%) than NBNC-HCCpatients (91.7%). This could be explained as most ofour NBNC-HCC patients were also suspected to havediabetes as depicted from their high fasting blood glucose

levels (data not shown) and this hyperglycaemic situa-tion can induce the generation of free radicals, whichmay ultimately influence the telomerase activity byincreasing oxidative stress (2426). These findings wereexciting and have not been reported earlier, particularlyin view of enhanced telomerase positivity in NBNC-HCCcases. However, a recent study showed that high telomer-ase activity is a poor prognostic marker in HCC, but themain drawback in this study is that it only includedHBV-related HCC cases (27). Nonetheless, these resultsare exciting and further studies with a large number ofHCC cases, especially NBNC-related HCCs, need to becarried out in this regard.

The expression of hTERT is rate limiting for telomer-

ase activity (28). In our study, hTERT mRNA expressionwas found in 81% of HCCs and in 11.8% of chronichepatitis cases. The correlation between telomeraseactivity and hTERTexpression was statistically significant(Po 0.0001). 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 significantlyshorter than that of chronic hepatitis tissues. The main

function of telomeres is to act as protective caps atthe ends of chromosomes, thus preventing end-to-endfusion of chromosomes and further chromosomal in-stability. Telomere shortening promotes chromosomalinstability during ageing and chronic diseases. It occursduring the course of chronic liver disease and is one of

the causes of hepatocarcinogenesis (14, 30). A range ofstudies in different human cancers demonstrate shor-tened telomeres in tumorous tissues (31). However,carcinogenesis is believed to be a multistage processdriven by genetic damage and epigenetic changes. Thus,a trend towards a shorter TL in cirrhotic HCC cases ascompared with non-cirrhotic HCC cases, which was seenin this study, can be explained by the fact that thesepatients have accumulated greater number of geneticalterations during the process of cell cycles and/orprogression of the disease. Moreover, the risk of HCCincreases sharply at the cirrhosis stage that is character-ized by hepatocyte telomere shortening, although thiswould have been more clear if we had included a pure

cirrhotic group without any evidence of HCC in ouranalysis. But, this was practically not possible as cirrhoticpatients were not amenable to any invasive procedures.Our data support the telomere hypothesis that chronicliver injury induces continual waves of liver destructionand regeneration, resulting in critical telomere short-ening, which in turn culminates in hepatocyte-replicativesenescence and ultimately in liver cirrhosis (32). Inhumans, few studies have described the shortening oftelomeres in cirrhosis induced by viral hepatitis (33, 34).A recent study by Zhanget al. (35) also found that TL inHCC tissues was not correlated with other clinicalparameters, 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 first to include an NBNC-HCCs groupalong with HBV- and HCV-related groups, and exam-ined whether or not the TL and telomerase activity isdifferent according to the aetiology of HCC, none ofwhich appear to differ between the different aetiologiesof HCC. We also did not find any correlation of TL withthe Okuda staging of HCC. As such, the length of atelomere in a cell type is partially determined genetically,but several environmental factors also have effects on TLincluding cumulative oxidative and inflammatory andpsychological stress (25).

More specifically, the mechanism of HBV-related HCCcan be mainly attributed to the integration of the viral

genome into the hepatocyte chromosomal DNA, therebyleading to rearrangement and instability of the host DNA.Recent investigations have shown that the telomerasegene is targeted for integration in different HBV-relatedHCCs, suggesting common pathways in HBV-relatedcarcinogenesis (36). While the involvement of HCV inhepatocarcinogenesis can be explained by involvement ofthe HCV core protein, leading to the overproduction ofoxidative stress, which yields genetic aberrations, modu-lation of cellular gene expressions and altered intracellular




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signal transductions, the combination of these alterationswould be hypothesized to promote the development ofHCC in HCV infection (37). However, the cause ofNBNC-HCC can be mainly attributed to the persistentexistence of diabetes and/or non-alcoholic steatohepatitisas an underlying ailment. In our chronic hepatitis cases,

the MTL was found to be 7.04 1.10 kb, which is slightlyshorter than that reported previously (7.30 1.40 and8.90 1.74 kb respectively) (14, 33). This may be becauseof the differences in age and the clinical stage of chronichepatitis patients.

A significant correlation of shorter telomeres withpositive telomerase activity was found in our study (Fig.8). This observation was interesting and has not beenreported earlier. Usually, when the length of a telomere islong enough for proliferation, telomerase activity sub-sides (38) but some mammalian cells, also without anytelomerase activity, are able to maintain the length oftheir telomeres for many population doublings (23), thusindicating the existence of one or more non-telomerase

mechanism(s) for telomere maintenance that have beentermed alternative lengthening of telomeres (ALT) (23,39). To date, clear evidence for ALT activity has only beenfound in some immortalized human cell lines and intelomerase-null mouse cell lines (40, 41). It seems likelythat understanding this form of telomere maintenance inHCC may have important implications for the diagnosisand treatment of HCC. Further studies are required toconfirm this form of telomere maintenance in HCC.

Acknowledgements

The authors acknowledge the financial support provided

by the Indian Council of Medical Research (ICMR) forthis study.Disclosures: None.

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Supporting information

Additional supporting information may be found in theonline version of this article:

Table S1. Telomerase activity, telomere length andhTERTmRNA expression in 58 hepatocellular carcinomacases.

Table S2. Telomerase activity, telomere length andhTERT mRNA expression in 17 chronic hepatitis cases.

Please note: Wiley-Blackwell is not responsible forthe content or functionality of any supporting materialssupplied by the authors. Any queries (other than missingmaterial) should be directed to the corresponding author

for the article.




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telomerase activity, telomere length and human telomerase reverse

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