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Eur J Cardiothorac Surg 2004;26:425-431
© 2004 Elsevier Science NL

Correlation between telomerase expression and terminal restriction fragment length ratio in non-small cell lung cancer—an adjusted measurement and its clinical significance

Chung Ping Hsua,b,c*, Jen Miawa,c, Sen Ei Shaia,c, Chih Yi Chena,c

a Division of Thoracic Surgery, Department of Surgery, Taichung Veterans General Hospital, Taichung 407, Taiwan, ROC
b School of Medicine, National Yang Ming University, Taipei, Taiwan, ROC
c School of Medicine, Chung Shang Medical University, Taichung, Taiwan, ROC

Received 29 January 2004; received in revised form 25 March 2004; accepted 5 April 2004.

* Corresponding author. Address: Division of Thoracic Surgery, Department of Surgery, Taichung Veterans General Hospital, 160, Sec. 3, Taichung-Kang Rd, Taichung 407, Taiwan, ROC. Tel.: +886-4-23592525x5050; fax: +886-4-23599715
e-mail: cliff{at}vghtc.gov.tw


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: To establish a new model for analyzing the correlation between the terminal restriction fragment (TRF) length and telomerase activity in non-small cell lung cancer (NSCLC) patients due to inconsistent results in previous reports. Methods: Between January 1999 and December 1999, 79 NSCLC patients were studied. The activity of telomerase was measured by telomeric repeat amplification protocol, and the telomere restriction fragment (TRF) length was measured by Telomere Length Assay Kit and Southern blotting. The correlation between expression of telomerase activity and the TRF length ratio (TRFLR) using the tumor-to-normal TRFLR (t/n TRFLR) was examined. Results: Positive telomerase activity was observed in 48 (60.8%) of the tumor tissue samples and in 5 (6.3%) of the normal tissue samples (P<0.0001). The mean TRF lengths were 5.32±1.53 kb in normal tissue samples and 3.95±1.92 kb in tumor tissue samples, respectively (P=0.0001). The 4-year cumulative survival rates of lower t/n TRFLR (less double equals75%) and higher t/n TRFLR (>75%) patients were 69.2 and 41.2%, respectively (P=0.0227). Independent prognostic factors include t/n TRFLR (P=0.014), T-status (P=0.027), and N-status (P=0.020) of the tumor. Conclusions: Our data suggest that there is a good correlation between the t/n TRFLR and expression of telomerase activity. A higher t/n TRFLR may indicate that the tumor regains its ability to repair the telomere lost and escapes the apoptosis scenario, which is subsequently reflected in poorer patient outcomes.

Key Words: Telomere • Telomerase • Terminal restriction fragment length • Lung cancer


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
Telomerase is a ribonucleoprotein, which synthesizes telomeric DNA located at the end of the chromosome. This enzyme is essential for the maintenance of the telomere's length that is characterized by its specific tandem repeat (5'-TTAGGG-3') of the human eukaryotes [1–3]. Human telomeres in somatic cells undergo progressive shortening with each replication cycle [3]. This shortening acts as a signal for replicative senescence and probably functions as a tumor suppressor mechanism in vivo. Activation of telomerase can bypass senescence and lead to immortalization. Normally, new telomeric repeats are added to the chromosomal end of the germline cells to maintain their stability and also preserve their full genomic information for the next generation [4]. Similarly, immortalized cell lines and more than 80% of the cancer cells can prevent the telomere from progressive shortening by up-regulation of telomerase, whereas the remainder maintains telomeres by a alternative lengthening of telomeres (ALT) mechanism [5]. These repairing processes may be regulated by a length-sensing feedback mechanism when the critical point is reached [6].

Telomerase is a holoenzyme that consists of a human telomerase reverse transcriptase (h-TERT), and a human telomerase RNA component (h-TERC), that acts as template for telomeric DNA synthesis [7,8]. Another protein that is associated with the holoenzyme is telomerase-associated protein (TP1), but recent studies have shown that it is not essential for telomerase activity in vivo [9]. In 1994, a sensitive polymerase chain reaction (PCR)-based technique, the telomeric repeat amplification protocol (TRAP) assay was developed for detection of telomerase activity. Our previous report had demonstrated good correlation between the expressions of h-TERT (not telomerase) and its associated genes such as c-Myc, TRF1 and TRF2 [10]. The link between the h-TERT expression, telomerase activation and telomere replication is not clear. We speculate that some other factors may play a role in allowing tumor cells to detect the chromosome end crisis, and initiate the process of telomerase expression. In this study, we used the TRAP assay to measure the telomerase activity in cancer and normal tissues of NSCLC patients. Subsequently, we used the Southern blotting method to measure the length of the telomere (TRF, terminal restriction fragment). The discrepancies between the cancer and normal tissues were compared, and their correlations with telomerase activity were analyzed. We also try to establish a new model for analyzing the correlation between the TRF length and telomerase activity in non-small cell lung cancer (NSCLC) patients due to inconsistent results in previous reports.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Patients and follow-up
Between January 1999 and December 1999, we included 79 cases of NSCLC (squamous cell carcinoma in 42, adenocarcinoma in 34, and large cell carcinoma in 3) who underwent surgical resection in this prospective study. None of the patients received pre-operative chemotherapy or radiotherapy. Whole body bone scan and liver sonography were performed for all of the patients to rule out distant metastasis. The tumor differentiation included well-differentiated carcinoma in 1, moderately differentiated carcinoma in 50, and poorly differentiated carcinoma in 28. Tumor staging was performed according to the AJCC (6th edition) criteria [11]. The p-TNM stages included stage I in 29, stage II in 11, stage III in 34, and stage IV in 5. The demographic data of the patients are listed in Table 1. All of the patients had been followed up to April 30, 2003.


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  		Table 1. Demographic data of the 79 NSCLC patients

 
2.2. Preparation of cell extracts
Twenty milligrams of frozen tissue samples were lysed with 200 µl lysis buffer and homogenized by polytron. Samples were then incubated in ice for 30 min and the lysate centrifuged at 16,000xg at 4 °C for 20 min. The supernatant was transferred to a fresh tube and the protein concentration was determined by the Bradford assay (Bio-Rad Protein Assay Kit, Bio-Rad Lab., Hercules, CA).

2.3. DNA isolation from tissues
Twenty-five milligrams of fresh frozen tissue was lysed with 800 µl lysis buffer containing 0.5% sodium dodecyl sulphate (SDS), 2 mM EDTA, 0.5 M NaCl, 10 mM MgCl2, 10 mM KCl and 10 mM Tris–HCl (pH 76), and digested with proteinase K at 50 µg/ml at 50 °C for at least 2 h. High molecular weight DNA was extracted with phenol/chloroform.

2.4. Assay for telomerase activity
The telomerase activities were measured by Southern blotting according to the results of TRAP assay in NSCLC tumor tissues as our previous report [10]. Telomerase activity was measured twice in independent experiments using 1–3 µg of total protein. Assays were performed using Telomerase PCR ELISA Kit (Boehringer Mannheim GmbH, Mannheim, Germany) including TRAP assay and detection by ELISA in two steps. In the first step, using TRAP, cell extracts were incubated with biotinylated telomerase substrate oligonucleotide (P1-TS) at 25 °C for 30 min, followed by 94 °C for 10 min to inactivate the telomerase. The extended products were amplified by PCR using Taq polymerase, the P1-TS, P2 primers and nucleotides. The PCR conditions were 33 cycles of 94 °C for 30 s on a DNA thermocycler (GeneAmp PCR System 9700, Perkin Elmer, Norwalk, CT, USA). In the second step, using the ELISA method, the amplified products were immobilized onto streptavidin-coated microtiter plates via biotin–streptavidin interaction, and then detected by anti-digoxigenin (DIG) antibody conjugated to peroxidase. After addition of the peroxidase substrate (3,3',5,5'-tetramethyl benzidine), the amount of TRAP products were determined by measurement of their absorbance at 450 nm (with a reference wavelength of 690 nm). Negative control reactions were performed by incubating cell extracts with 1 µg/µl RNase for 20 min at 37 °C. The results were interpreted as negative, 1+, 2+, and 3+ when the OD (optic density) values were <0.2, 0.2–1, 1–2, and >2, respectively.

In addition, to confirm the ELISA results, amplified products were systemically run on 15% non-denaturing polyacrylamide gel. After transferring the PCR products onto a positively charged nylon membrane, Southern blotting was performed by the semi-dry electrophoretic blotting instrument (Multiphore II NovaBlot Unit, Amersham Pharmacia Biotech, Buckinghamshire, UK). The membrane was then incubated with a streptavidin alkaline phosphatase conjugate (1:5000 dilute in blocking solution), and after rinsing, blotted products were visualized by Biotin Luminescence Detection Kit (Boehringer Mannheim, Mannheim, Germany). In addition, all telomerase-negative tumor specimens were re-checked by additional TRAP assay using a 150 bp internal telomerase assay standard to exclude the possibility of Taq DNA polymerase inhibition in the tumor extracts [12].

2.5. Terminal restriction fragment (TRF) length measurement
TRF length measurement was performed using TeloTAGGG Telomere Length Assay Kit (Roche, Mannhein, Germany). Eight micrograms of genomic DNA was digested with each 30 U Hinf 1/Rsa l at 37 °C for 16 h. The resulting fragments were fractionated by electrophoresis on 0.8% agarose gel and transferred to nylon membrane using Southern blotting. After transfer, the transfer DNA was fixed on the membrane by UV-crosslinking (120 mJ). The membrane was first pre-hybridized at 42 °C for 30 min and then hybridized with telomere-specific DIG-labeled probe at 42 °C for 3 h. After washing the membrane in 2x SSC, membrane was incubated with anti-DIG-alkaline phosphatase (1:5000 dilute in blocking solution). Finally, the immobilized telomere probe was visualized by alkaline phosphatase metabolizing CDP-Star, a highly sensitive chemiluminescence substrate. The membrane was then exposed to X-ray film, and the average TRF length was determined by comparing the signals relative to a molecular weight standard (using BIO-PROFIL Bio-1D Software, Version 99, Vilber Lourmat, France), and the mean of three measured TRF lengths deducted by 2.5 kb was used as the presented telomere length [13,14]. Furthermore, the TRF length ratio (TRFLR) was defined as the ratio between the length of tumor tissue TRF (t-TRF) and their paired normal tissue TRF (n-TRF) from the same patient.

2.6. Statistical analysis
Cumulative survival curves were calculated and drawn using the Kaplan–Meier method and subgroups were compared by the log-rank statistic. Multivariate analyses were performed using the Cox proportional hazards model. All probabilities were two-tailed, with a P-value less than 0.05 regards as statistically significant. The statistical calculations were conducted with SPSS software (v10.5, SPSS Inc., Chicago, Ill).


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Expression of telomerase activity
Positive telomerase activities were observed in 48 of 79 (60.8%) tumor tissue samples, and 5 of 79 (6.3%) normal tissue samples, respectively. The distribution of telomerase activity expression is listed in Table 2 according to patients' age, sex, tumor cell type, tumor differentiation, tumor TNM status, and stages. Higher percentage of telomerase expression is observed in patients with larger tumor burden (T-status) (P=0.0265) and later tumor stages (P=0.0294).


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  		Table 2. Expression of telomerase activity according to the patient's characteristics in 79 NSCLCs

 
3.2. Terminal restriction fragment length
The mean terminal restriction fragment (TRF) lengths were 5.32±1.53 kb in normal tissue samples and 3.95±1.92 kb in tumor tissue samples, respectively (P<0.0001). The mean TRF lengths were 3.87±1.96 kb in telomerase-positive tumor tissue samples and 4.06±1.88 kb in telomerase-negative tumor tissue samples, respectively (P=0.136). The mean TRF lengths were 4.00±2.23 kb in stage I tumor, 4.46±1.84 kb in stage II tumor, 3.61±1.54 kb in stage III tumor, and 4.79±2.21 kb in stage IV tumor, respectively (P=0.321). In an overall comparison, as shown in Table 3, the TRF length did not demonstrate any significant relationship to the TNM stages. The telomeric repeat signals were demonstrated as smear on gel as shown in Fig. 1 .


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  		Table 3. TRF length according to the expression of telomerase activity and TNM stages

 


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  		Fig. 1. Representative TRF length by Southern blotting analysis in paired normal (N) and tumor (T) tissues of NSCLC. TRF length (T/N) in patients 1, 4, 18 and 38 were 5.66/6.55, 4.67/5.66, 4.96/5.56, and 6.74/7.56 kbs, respectively.

 
3.3. The t/n TRFLR
The overall mean tumor-to-normal TRFLR (t/n TRFLR) was 73.4±24.2% (42.0–106.9%). By setting a cut-off value of 75%, the patients can be divided into two groups. The first group (n=39) with a t/n TRFLR less double equals75% had a mean ratio value of 56±17% (stage I in 16, stage II in 3, stage III in 19, and stage IV in 1); the second group (n=40) with a t/n TRFLR >75% had a mean ratio value of 89±18% (stage I in 13, stage II in 8, stage III in 15, and stage IV in 4). The distribution of t/n TRFLR in stage I–IV tumors were 0.72±0.24, 0.87±0.32, 0.69±0.22, and 0.73±0.10, respectively. The t/n TRFLR did not correlate well with the tumor stages (P=0.189). Correlation between the t/n TRFLR and the expression of telomerase activity are shown in Fig. 2 . A negative linear correlation was found (r=–0.298, P=0.012).



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  		Fig. 2. Correlation between expression of telomerase activity in tumor tissues and t/n TRFLR were examined by XY chart, and a linear correlation was established.

 
3.4. Effect on survival rates
The influence of telomerase activity expression was evaluated by cumulative survival period. As shown in Fig. 3 , the 4-year cumulative survival rates of telomerase-positive and telomerase-negative patients were 53.4 and 58.1%, respectively (P=0.9071). When survival analyses were performed based on the change of TRF length in the tumor tissues, a cut-off value of 75% demonstrated significant survival difference. As shown in Fig. 4 , the 4-year cumulative survival rates of lower t/n TRFLR (less double equals75%) and higher t/n TRFLR (>75%) patients were 69.2 and 41.3%, respectively (P=0.0227). Multivariate survival analysis using Cox proportional hazards model revealed independent prognostic factors includes t/n TRFLR (P=0.014), T-status (P=0.027), and N-status (P=0.020) of the tumor (see Table 4).



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  		Fig. 3. Cumulative survival rates in 79 NSCLC patients according to the expression of telomerase activity in tumor tissues.

 


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  		Fig. 4. Cumulative survival rates according to the t/n TRFLR in 79 NSCLC patients.

 

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  		Table 4. Multivariate survival analysis for Cox proportional hazards model

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Expression of the telomerase activity in tumor tissues has been repeatedly reported in several malignancies [15–18]. The reported incidences of telomerase expression in NSCLCs were between 60 and 82% [19–21]. Expression rate in our data was 61% which is similar to rates reported in other studies. Most of the reported series failed to demonstrate significant associations between the expression of telomerase activity and tumor characteristics, such as cell differentiation, pathological grading, and tumor stages [19–21]. Our data also demonstrated the similar findings, and we believe this may indicate poor association between the telomerase activity and malignant potential of the tumor. As shown in our data, expression of the telomerase activity occurred in every tumor stage. We speculate that other factors play a role in allowing tumor cells to detect the chromosome end crisis. Previous reports concerning the impact of telomerase activity expression on prognosis of NSCLC are controversial [19,21,22]. Our data did not demonstrate significant survival differences based on the expression of telomerase activity. This might be due to small sample size in this study.

Progressive TRF length shortening has been demonstrated in different cancer cell lines [19]. The TRF length of 79 paired tissues was measured by Southern hybridization method using the TeloTAGGG Telomere Length Assay Kit. The TRF length of the tumor tissues was shorter than their paired normal lung tissues (P=0.0001). Overall comparison of the TRF length in different tumor stages did not reach a significant level (P=0.321). Our data indicate that a more frequent cellular division in advanced staged tumor tissue may lead to a more significant loss of their telomeric ends. Table 3 shows that the mean TRF length was shorter in the telomerase(+) tumors, which may imply a negative feedback stimulation of the telomerase activity in tumor tissues. By doing so, the tumors become immortal, and also maintain their malignant characteristics such as local invasiveness and distant metastasis for persistent disease progression. The controlling factors involved in expression of h-TERT gene, telomerase activity, and maintenance of telomere length are complex. Our previous study had demonstrated high expression rates of the h-TERT, h-TERC, TP1, c-Myc, TRF1 and TRF2 genes (66.6, 92.3, 100.0, 91.0, 74.4 and 83.3%, respectively) in the tumor tissues. In addition to the tumor tissue itself, higher telomerase expression rates were observed in h-TERT(+), TRF1(+) tumor tissues compared to their normal counterparts [10]. We also found high concordance in expression of the h-TERT gene (not telomerase activity) and its associated genes [10]. These data suggest that TRF1 and TRF2 expression, working together with the dynamic changes of t/n TRFLR, may activate h-TERT expression in telomere repair. However, the detail mechanisms how these factors interact with each other deserve further clarification.

In previous reports, comparison of the TRF lengths and their impact on prognosis were performed either based on the tissue types (tumor vs. normal) or based on the tumor stages [16,17,23]. One report showed a significant (2SD) alteration of TRF length indicated shortened survival times in NSCLC [22]. One major handicap of the above methods is that the tumor's age at diagnosis is not considered. This means at the very beginning of tumorgenesis, cellular ages are different in individual tumor tissue; subsequently, the TRF length should be different. To take this consideration into account, we analyzed the t/n TRFLR using the t-TRF (tumor tissue TRF length) divided by the n-TRF (paired normal tissue TRF length). Our hypothesis is based on a cellular age-adjusted concept, avoiding inconsistency between the t-TRF length and the expression of telomerase activity. By a series examination, we found that a cut-off value of 75% for the t/n TRFLR can be used for prognosis evaluation. Our data suggest that when the TRF length is decreased to a certain level, the telomerase activity can be elicited. This hypothesis may explain the good correlation between the t/n TRFLR and the expression of telomerase activity as shown in Fig. 4. Unfortunately, telomerase expression is not the only route for telomere repair. ALT pathway itself or coexists with telomerase also contributes to repair of the chromosome ends [24]. This may partially explain why telomerase expression itself is not well correlated with prognosis in our patients.

Our data indicate that the presence of telomerase activity itself is not a good predictor of survival. However, progressive TRF length shortening (reflected by t/n TRFLR) of the tumor may subsequently activate the telomerase activity of the tumor tissue by a negative feedback mechanism. This will allow the tumor to escape the apoptosis scenario, and eventually reflects by a poorer survival.


   Acknowledgments
 
The authors thank Ms L.W. Lee and Ms H.C. Ho (Biostatistics Task Force of Taichung Veterans General Hospital) for their assistance in tissue preparation, data recording, and statistical analysis. This study was supported by grants from the Professor K.S. Lu Lung Cancer Foundation, Taipei, Taiwan, ROC.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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