Japanese Journal of Clinical Oncology Advance Access originally published online on October 23, 2006
Japanese Journal of Clinical Oncology 2007 37(1):16-22; doi:10.1093/jjco/hyl118
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© 2006 Foundation for Promotion of Cancer Research
Telomere Length, Telomerase Activity, and Expressions of Human Telomerase mRNA Component (hTERC) and Human Telomerase Reverse Transcriptase (hTERT) mRNA in Pulmonary Neuroendocrine Tumors
1 Department of Pathology and Laboratory Medicine
2 Department of Surgery, National Defense Medical College, Tokorozawa, Saitama
3 Department of Pathology, Kitasato University, Sagamihara, Kanagawa
4 Pathology Division, Shizuoka Cancer Center Hospital and Research Institute, Sunto-gun, Shizuoka
5 Department of Pathology, National Tokyo Hospital, Kiyose, Tokyo
6 Division of Diagnostic Pathology, Keio University School of Medicine, Tokyo, Japan
7 Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, NY
8 Armed Forces Institute of Pathology, Washington DC, USA
For reprints and all correspondence: Kuniaki Nakanishi, Department of Pathology and Laboratory Medicine, National Defense Medical College, Tokorozawa 359-8513, Japan. E-mail: nknsknak{at}ndmc.ac.jp
Received May 15, 2006; accepted August 18, 2006
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Abstract |
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 TOP Abstract INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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BACKGROUND: Telomeres are important for chromosome structure and function, protecting them against degradation. However, few studies have examined telomeres in pulmonary neuroendocrine (NE) tumors.
METHODS: We investigated deparaffinized sections obtained from 70 primary NE lung tumors [34 typical carcinoids (TCs), 10 atypical carcinoids (ACs), 16 large cell neuroendocrine carcinoma (LCNECs) and 10 small cell lung carcinomas (SCLCs)].
RESULTS: Positive expressions of human telomerase mRNA component (hTERC) and human telomerase reverse transcriptase (hTERT) mRNA were recognized, respectively, in 58% and 74% of TCs, and in 100% and 100% of ACs, LCNECs and SCLCs. Alteration of telomere length was greater in both LCNECs and SCLCs than in TCs. Telomerase activity was detected in LCNECs, but not in TCs. By the reverse-transcriptase polymerase chain reaction (RT-PCR), hTERC mRNA was detected in 100% of LCNECs and TCs examined, while hTERT mRNA was detected in 67% of LCNECs, but not at all in TCs.
CONCLUSIONS: These results suggest that alterations in telomere length, telomerase activity, and the expression of hTERT mRNA may (i) play roles in pathogenesis in pulmonary neuroendocrine tumors, and (ii) be a useful tool for differential diagnosis between TCs and LCNECs.
Key Words: Telomere • telomerase • human telomerase mRNA component (hTERC) • telomerase reverse transcriptase (hTERT) • neuroendocrine tumors
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INTRODUCTION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Telomeres are important for the structure and function of chromosomes, because they protect chromosomes from degradation and also appear to contribute to the attachment of chromosome ends to the nuclear envelope (1,2). Telomeres lose a portion of their non-coding repetitive DNA sequence with each cell division. When telomeres reach a critical length, cells lose their proliferating potential, resulting in cellular senescence. Telomerase is the ribonucleoprotein enzyme that synthesizes the strand of telomeric DNA, and it thereby compensates for the loss of this DNA during each cell division (2). Despite the observation that some immortalized cell lines manifest stable telomeres without evidence of telomerase activity, telomerase-dependent telomere elongation seems to be the most important mechanism for maintaining telomere length both in vivo and in vitro.
During the last decade, two major subunits of human telomerase, namely, human telomerase RNA (hTERC) and human telomerase reverse transcriptase (hTERT), have been identified (3–6). Of these, hTERC, cloned as the template RNA for telomerase, is predominantly seen in fetal tissues, while hTERT contains the reverse transcriptase that catalyzes this reaction. Several studies have demonstrated that hTERC and hTERT form the minimum complex needed for telomerase activity (3–6).
In humans, shortening of telomere length during aging in vivo has been observed in most normal somatic cells, but not in sperm DNA (1,2), and moreover, telomerase is active both during embryonic development and in adult germ-line tissues, but not in most adult somatic tissues (2,7,8). Although several studies have investigated telomere length and telomerase activity in pulmonary tumors (9–17), few have examined these variables in pulmonary neuroendocrine tumors (18–20).
Recent attempts at classification of pulmonary neuroendocrine tumors have identified four categories of tumors: low grade typical carcinoid (TC), intermediate grade atypical carcinoid (AC), high grade large cell neuroendocrine carcinoma (LCNEC), and small cell lung carcinoma (SCLC). LCNEC is a category that was newly added in 1999 to the World Health Organization (WHO) Classification of Lung Tumours, and it was maintained as a category in the 2004 WHO classification (21). Even pathologists sometimes find difficulty in distinguishing these different types of pulmonary neuroendocrine tumors (22), and therefore clinical, pathologic and molecular characteristics of these tumors are not fully understood. In the current study, we tried to clarify these points: (i) by employing an in situ hybridization approach using hTERC and hTERT mRNA probes to examine deparaffinized tissue sections obtained from 67 primary pulmonary neuroendocrine tumors, (ii) by measuring telomere length and telomerase activity using the polymerase chain reaction (PCR)-based telomerase repeat amplification protocol (TRAP) assay, and (iii) by using the reverse-transcription polymerase chain reaction (RT–PCR) to examine the expressions of hTERC and hTERT mRNA in frozen tissues. The study aimed to determine whether changes in telomere length, telomerase activity and the expressions of hTERC and hTERT mRNA might be good markers to help distinguish the four histological subtypes of pulmonary neuroendocrine tumors.
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MATERIALS AND METHODS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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The material used comprised 70 surgically resected specimens from patients with primary pulmonary neuroendocrine tumors (34 TCs, 10 ACs, 16 LCNECs and 10 SCLCs). These specimens had been obtained at the National Defense Medical College Hospital, Keio University Hospital, Kitasato University Hospital, Tokyo Hospital and the Armed Forces Institute of Pathology between 1978 and 2001. Histological slides were reviewed, and the cases were divided into the following categories according to the standardized histopathological criteria described in the WHO classification (21). The frozen specimens were embedded in OTC compound materials, and stored at –80°C.
In Situ Hybridization
Samples (19 TCs, 10 ACs, 9 LCNECs and 5 SCLCs for hTERC, and 34 TCs, 10 ACs, 16 LCNECs and 10 SCLCs for hTERT mRNA) were formalin-fixed and paraffin-embedded, and were examined by in situ hybridization for hTERC and hTERT mRNA. In situ hybridization was performed essentially as previously described (23). Briefly, sections were treated with 0.2 N HCl for 20 min, then incubated in 2x standard saline citrate (SSC) for 10 min at 37°C, and finally incubated in 5 µg/ml proteinase K (Merck, Darmstadt, Germany; 1 µg/ml in phosphate-buffered saline [PBS]) for 10 min at 37°C. Sections were subsequently post-fixed in 4% paraformaldehyde in PBS for 5 min, washed with 0.2 % glycine for 10 min, then incubated in 0.1 mol/l triethanolamine buffer, pH 8.0, containing 0.25% (vol./vol.) acetic anhydride for 10 min to prevent non-specific binding caused by oxidation of the tissue. Finally, they were washed with 95% ethanol and dried. Hybridization was carried out overnight at 42°C in 50% (vol./vol.) deionized formamide, 5x Denhardt solution, 5% (wt/vol.) dextran sulfate, 2x SSC, 0.3 mg/ml salmon sperm DNA, 5 mM ethylenediaminetetraacetic acid, and 10 ng/ml biotin-labeled sense and antisense oligonucleotide probes for hTERC and hTERT (100 pmol per slide). For the probes, we chose portions of hTERC and hTERT mRNA, as indicated by Feng et al. (6) and Nakamura et al. (3). These probes were prepared with 48 (123–170) and 60 (1807–1866) oligonucleotides, respectively. The sense probe was used as a negative control. After incubation, the sections were washed sequentially, twice with 2x saline-sodium citrate (SSC) at 42°C for 5 min, once with 2x SSC at room temperature (RT) for 10 min, and once with 0.2x SSC at RT for 10 min. Signal amplification was performed using an ABC kit (DAKO, Carpinteria, CA). hTERC and hTERT mRNA were detected using BCIP (5-bromo-4-chloro-3-indolyl phosphate; Sigma Chemical, St Louis, MO). For our analysis of reactivity, those tumors in which the stained tumor cells made up >10% of the tumor were graded as positive.
DNA Extraction and Analysis of Telomere Length
Fresh frozen samples from nine specimens (two TCs, three LCNECs and four SCLCs) were incubated in cell lysis solution (0.04% Proteinase K, 10 mM Tris-Hcl (pH 8.0), 1 mM EDTA, and 1% Tween-20) at 37°C for 16 h, essentially as previously described (23). The DNA was extracted using a DNA extraction kit (Gentra Systems, Minneapolis, MN). Analysis for telomere length was performed essentially as previously described (8). Briefly, 1 µg of DNA digested with Hinf I and Rsa I at 37°C for 2 h was loaded onto 0.8% agarose gels, and separated by electrophoresis at 50 V (5 V/cm2) for 3 h. The 1-kb DNA ladder (GIBCO BRL, Karlsruhe, Germany) served as a molecular weight marker. The gels were denatured and vacuum-blotted to a positively charged nylon membrane (Imobilon N; Millipore, Bedford, MA). After cross-linking the DNA to the membrane with the aid of UV light (120 mJ), the filters were hybridized to a digoxigenin-labeled (TTAGGG)7 telomeric probe (Roche Diagnostics GmbH, Mannheim, Germany). Hybridization was performed at 42°C for 3 h. Blots were washed twice for 5 min in 2x SDS at 50°C. Detection of the DNA fragments that had hybridized to the digoxigenin-labeled probes was performed by chemiluminescence, the manufacturer's instructions being followed exactly (Roche Diagnostics GmbH). Softex films (Fuji, Tokyo, Japan) were exposed within the linear range of the chemiluminescence reaction for 1–5 min. Exposed films were scanned and analyzed using NIH image 1.3.1. Mean lengths were defined as 
(ODi)/(ODi/Li) where ODi is the densitometer output over interval I, while Li is the kilobase size at the middle of interval i.
Telomerase Repeat Amplification Protocol Assay
Telomerase activity was examined in fresh frozen samples from 11 specimens {two TCs, three LCNECs and six non-SCLCs [four large cell carcinomas (LaCs), one squamous cell carcinoma (SqC), and one combined cancer (adenocarcinoma plus large cell carcinoma; AdC + LaC)]} using a TRAP-eze detection kit (Intergen, Manhattanville, NY) and Fluor Imager system (Molecular Dynamics, Sunnyvale, CA) (23). The TRAP assay procedure was performed according to the manufacturer's instructions. Frozen samples of both tumors and non-neoplastic tissues were homogenized and suspended in 20–50 µl of CHAPS {i.e. 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate lysis buffer}, then placed on ice for 30 min, and finally centrifuged at 13 500 rpm for 30 min at 4°C. The supernatants were collected and total protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA). Briefly, 2 µl of the cell extract (equivalent to 3 µg of protein) was added to 48 µl of a reaction solution consisting of 1x TRAP buffer (Intergen), 1x standard deoxynucleotide triphosphate mix ([dNTPs]; Intergen), sense primer 5'-AATCCGTCGAGCAGAGTT-3', TRAP primer mix containing a reverse primer and a 36-bp internal standard (Intergen), 2 U of Taq polymerase (Boehringer Mannheim, Germany) and distilled water. TSR8 (Intergen) was used as a positive external control. The mixture was incubated at 30°C for 30 min. The polymerase chain reaction (PCR) conditions were 30 cycles at 94°C for 30 s and 60°C for 30 s. The PCR products were electrophoresed on a 12.5% polyacrylamide gel, visualized with the aid of SYBR Green I Nucleic Acid Gel Stain (Takara Shuzo, Kyoto, Japan), and measured using the Fluor Imager system. The data were analyzed by means of the NIH image system (National Institute of Health, Bethesda, MD). Activity in the TRAP assay was detected as a ladder of products with six base increments (starting at 50 nucleotides) and the relative telomere activity was calculated semiquantitatively. To this end, the total product generated ([TPG]; units/micrograms protein) was defined as follows: first, in samples of lung carcinoma the ratio of telomerase products (starting from 50 bp) to their short internal standard (36 bp) was calculated. Then, this ratio was corrected by means of an external standard (TSR8 cells; Intergen). We considered the reaction to be positive when the telomerase ladder was confirmed and the TPG was positive.
RT–PCR
Total mRNAs were obtained from three normal lung tissues, one TC, three LCNECs and seven non-SCLCs (four LaCs, one AdC, one SqC and one AdC + LaC). The total RNA was isolated using acid guanidinium isothiocyanate-phenol-chloroform extraction and ethanol precipitation. RT–PCR was performed using an amplification reagent kit (TaqMan EZRT-PCR kit; Applied Biosystems, Alameda, CA) with hTERC, hTERT mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers as follows: hTERC, 5'-GAAGAGGAACGGAGCGAGTC-3' (sense), 5'-AAAAAGCGGAAGACGGGAG-3' (antisense); hTERT mRNA, 5'-CGGCTTTTGTTCAGATGCC-3' (sense), 5'-AGCACACATGCGTGAAACCT-3' (antisense); GAPDH, 5'-GAAGGTGAAGGTCGGAGTC-3' (sense), 5'-GAAGATGGTGATGGGATTTC-3' (antisense). Primers were synthesized using an automated DNA synthesizer. The reaction master mix was prepared according to the manufacturer's protocol to give final concentrations of 1x reaction buffer, 300 µM dATP, 300 µM dCTP, 300 µM dGTP, 600 µM dUTP, 3 mM Mn(OAc)2, 0.1 unit/µl rTth DNA polymerase, 0.01 unit/µl AmpErase UNG, 900 nM primers and 200 nM TaqMan probe. To perform PCR, the reverse-transcription reaction was incubated at 60°C for 30 min, followed by incubation at 95°C for 5 min to deactivate AmpErase UNG. PCR was performed using 40 amplification cycles at 95°C for 20 s and at 60°C for 1 min using an ABI PRISM 9600 Sequence detector (Applied Biosystems). PCR products were separated by electrophoresis in a 3% agarose gel and stained with ethidium bromide.
Statistical Analysis
The results are expressed as mean ± standard error. Comparisons between two values were made using a Student's t-test, Mann–Whitney test, or Kruskal–Wallis test, as appropriate. Differences were considered significant at P < 0.05.
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RESULTS |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Expressions of hTERC and hTERT mRNAs
Expression of hTERC mRNA was confined to the cytoplasm of tumor cells (Fig. 1). Its expression varied from weak to strong. In the normal lung, a strong hTERC expression was detected in the serous cells of the bronchial glands and intrapulmonary lymphoid follicles, but it was barely detected at all in the non-ciliated cells of bronchioles or in alveolar type II cells. Sometimes it was detected in hyperplastic non-ciliated cells of bronchioles and in hyperplastic alveolar type II cells. A positive hTERC expression was recognized in 58% of 19 TCs, and in all 10 ACs, all nine LCNECs and all five SCLCs examined. With regard to the heterogeneity of expression, hTERC mRNA was detected as a diffuse pattern in AC, LCNEC and SCLC, while it was detected strongly in peripheral lesions of TC. Statistically, the differences between TCs and ACs, and between TCs and LCNECs, were significant (P = 0.016 and P = 0.021, respectively).
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Expression of hTERT mRNA was confined to the cytoplasm of tumor cells (Fig. 1). Its expression varied from weak to strong. In the normal lung, the pattern of expression of hTERT mRNA was essentially the same as that of hTERC. That is to say, it was (i) strongly represented in the serous cells of the bronchial glands and intrapulmonary lymphoid follicles, (ii) barely detected at all in non-ciliated cells of bronchioles or in alveolar type II cells, and (iii) sometimes detected in hyperplastic non-ciliated cells of bronchioles and in hyperplastic alveolar type II cells, with alveolar type I cells always being negative. A positive hTERT expression was recognized in 74% of 34 TCs, in all 11 ACs, all 16 LCNECs and all 10 SCLCs. With regard to the heterogeneity of expression, hTERC mRNA was detected as a diffuse pattern in AC, LCNEC and SCLC, while it was detected strongly in peripheral lesions of TC. Statistically, the difference between TCs and LCNECs was significant (P = 0.023).
Telomere Length
Data relating to telomere length were obtained from all specimens examined (Fig. 2). The mean telomere length in normal adjacent lung tissues was 10.9 ± 0.6 kbp (mean ± SE), while for two TCs, three LCNECs and three SCLCs the values were 8.7 ± 0.8 kbp, 7.7 ± 1.4 kbp, and 5.8 ± 1.2 kbp, respectively. When the values obtained for telomere length in TCs, LCNECs and SCLCs were compared with that obtained for paired normal adjacent lung tissues in each patient, we arbitrarily defined the telomere as reduced or elongated if the telomere length in tumor tissues was, respectively, shorter that 80% or longer than 120% of that of the normal adjacent lung tissues. Telomere length analysis revealed that two LCNECs and two SCLCs exhibited reduction, while one LCNEC exhibited elongation. Thus, three of three LCNECs and two of three SCLCs showed alterations in telomere length, but the two TCs were normal.
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Telomerase Activity
Telomerase activity was detected in neither of the two TCs examined, but in all of three LCNECs (mean ± SE; 4.2 ± 2.3) and in all of six non-SCLCs (four LaCs, one SqC and one AdC + LaC) (4.6 ± 1.0) (Fig. 3). Telomerase activity was significantly greater in LCNECs than in TCs (P < 0.05).
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RT–PCR
hTERC mRNA was detected on the gel as one band of 301 bp in all samples examined [three normal tissues, one TC, three LCNECs and seven non-SCLCs (four LaCs, one AdC, one SqC, and one AdC + LaC)], although the strength of the expression varied (Fig. 4). hTERT mRNA was detected on the gel as one band of 301 bp in two non-SCLCs and two LCNECs, but in no normal tissues or TCs.
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DISCUSSION |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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Although several studies have investigated telomere length and telomerase activity in pulmonary tumors (8–16), only one report [by Zaffaroni et al. (20)] has focused specifically on the quantitative and qualitative changes (including those in the expressions of hTERC and hTERT mRNA, telomere length and telomerase activity) occurring in pulmonary neuroendocrine tumors. The major findings made in the present study were that the positive expression of hTERT mRNA (detected using in situ hybridization and RT–PCR), the alteration of telomere length and the level of telomerase activity were all significantly greater in LCNECs than in TCs. These results obtained using in situ hybridization and RT–PCR for hTERC and hTERT mRNA are similar to those obtained using RT–PCR described by Zaffaroni et al. (20), and thus we suggest that the three variables above (the expression of hTERT mRNA, the alteration of telomere length and the level of telomerase activity) may together represent a useful tool for differential diagnosis between TCs and LCNECs. hTERC mRNA expression was significantly higher in both AC and LCNEC than in TC. While differential diagnosis between TC and LCNEC is not a challenge diagnostically, separating TC from AC may be problematic, and hTERC may be helpful in making this distinction.
It is well known that in a given population of cells, telomere length is determined by several factors, including cell division, telomerase activity, and telomeric associations and fusions (1,2). In lung cancers, telomere lengths have been found to vary widely in several studies (15–17), implying that each individual cancer has its own characteristic equilibrium between proliferative telomere shortening and its restoration by telomerase activity. Several investigators have reported that a high telomerase activity within cells can be interpreted as representing compensation for the shortening of telomere length resulting from uncontrolled proliferation, the consequence being a stabilized telomere length that is a characteristic of a given cell (1,2,4,5,25). In the present study, an alteration of telomere length was detected in 100% of LCNECs and in 67% of SCLCs, but in neither of two TCs. Admittedly, the samples we examined were from only a few cases, but these results suggest that detecting alterations of telomere length may be useful for differential diagnosis between TCs and LCNECs. The very recent study by Zaffaroni et al. revealed that telomeres were significantly longer in telomerase-negative carcinoids than in telomerase-positive carcinoids (20). Our results in TCs support those data.
In the present study, telomerase activity was detected in all three LCNECs and all seven non-SCLCs examined, but in neither of two TCs. This incidence of telomerase activity in TCs and LCNECs accords well with the figures for its incidence in pulmonary neuroendocrine tumors obtained in three previous studies performed using the TRAP assay or TRAP-ELISA (17,18,20). In one of those studies, Gomez-Roman et al. (17)—who examined three TCs, three ACs, four LCNECs and two SCLCs by TRAP-ELISA—reported that telomerase activity levels were similar between TCs and non-neoplastic lung tissues, but higher in ACs, LCNECs and SCLCs than in TCs. Zaffaroni et al. (18)—who examined 15 TCs, eight LCNECs and 15 SCLCs using the TRAP assay—detected a positive TRAP signal in 7% of TCs, 87% of LCNECs and 93% of SCLCs. Zaffaroni et al. (20)—who examined 44 TCs, 15 LCNECs and 27 SCLCs using the TRAP assay—detected a positive TRAP signal in 14% of TCs, 87% of LCNECs and 92% of SCLCs. To judge from the above data, telomerase activation may play an important role at a critical step determining malignant potential in pulmonary neuroendocrine tumors.
It has been shown that telomerase activity is absent from most normal tissues (25) and that hTERT expression correlates closely with telomerase activity both in vitro and in vivo (3,26). For example, hTERT mRNA expression is observed at high levels in cancer cell lines, but not in telomerase-negative cell lines (5,27). Therefore, hTERT transcription may play a part in the mechanism by which telomerase regulation is achieved. The expression of hTERT mRNA has been said to be more important than hTERC expression in the regulation of telomerase activity (28,29). Moreover, in several studies telomerase activity was detected during the early phase of carcinogenesis (viz. intestinal metaplasia to adenoma in the stomach, dysplasia to carcinoma in situ in the uterine cervix and intrapulmonary tract) (30–32). Those authors suggested that hTERT mRNA expression is upregulated and that telomerase activation is a critical step during tumor carcinogenesis, and that both can be recognized at an early stage in the oncogenic process. In pulmonary neuroendocrine tumors, Sarvesvaran et al. detected hTERC expression in 59% of 29 TCs, 57% of eight ACs and 98% of 62 SCLCs using in situ hybridization (33). Zaffaroni et al. (20)—who examined hTERT mRNA expression in 24 TCs, 12 LCNECs and 16 SCLCs using RT–PCR—detected it in 21% of TCs, 67% of LCNECs and 84% of SCLCs. In our in situ hybridization examination, expressions of hTERC and hTERT mRNA were detected in 58% and 74% of TCs, respectively, but in as many as 100% and 100% of ACs, LCNECs and SCLCs, findings similar to those of Sarvesvaran et al. (33), except in the case of ACs. In our RT–PCR, however, hTERC expression was detected in all of the TCs and LCNECs, while hTERT mRNA was detected in two of three LCNECs, but in none of the TCs, findings similar to those of Zaffaroni et al. (20). On this basis, our results support the idea that upregulation of hTERT mRNA may play an important role at a critical step determining malignant potential, as well as serving to upregulate telomerase activity. At present, the reason for the discrepancy concerning hTERC expression in TCs (viz. between 58% by in situ hybridization and 100% by RT–PCR) is unclear. However, one possible factor is that the sensitivities of the hTERC primers may be different and/or that the hTERC may be localized to foci within tumors. More recently, Zaffaroni et al. (20), who analyzed the alternative splicing of hTERT, detected full-length hTERT mRNA in five of five TRAP-positive and 10 of 19 TRAP-negative CTs. They pointed out the importance of splice variants in determining TRAP activity in CT, because transcription of the hTERT gene is a major regulatory step in the process of telomerase reactivation and alternative splicing of hTERT transcripts may be involved in the regulation of telomerase activity.
In conclusion, our study suggests that certain characteristic features (namely, hTERT mRNA expression, telomere length and telomerase activity) may differ between TCs and LCNECs. Recently, telomerase-specific DNA-binding proteins have been put forward as additional candidates for the molecules modifying telomerase activity (34,35). To function properly, human telomeres require both TRF1 (telomeric repeat binding factor-1) and TRF2 (since TRF1 negatively regulates the maintenance of telomere length, while TRF2 protects telomeres from end-to-end fusion) (34,35). Further studies will be needed to examine the alterations in telomere binding proteins in pulmonary neuroendocrine tumors.
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Conflict of interest statement |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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None declared.
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Acknowledgment |
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The authors are indebted to Sadayuki Hiroi and Susumu Tominaga for their expert technical support.
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References |
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TOPAbstractINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONConflict of interest statementReferences |
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