Expression of Mrnas for Telomeric Repeat Binding Factor (TRF)-1 and TRF2 in Atypical Adenomatous Hyperplasia and Adenocarcinoma of the Lung
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Vol. 9, 1105–1111, March 2003 Clinical Cancer Research 1105 Expression of mRNAs for Telomeric Repeat Binding Factor (TRF)-1 and TRF2 in Atypical Adenomatous Hyperplasia and Adenocarcinoma of the Lung Kuniaki Nakanishi, Toshiaki Kawai,1 positive expressions of TERF2 mRNA among low-grade Fumiyuki Kumaki, Sadayuki Hiroi, Makio Mukai, AAH, high-grade AAH, and BAC. Eiji Ikeda, Catherine Elaine Koering, and Conclusions: These results are consistent with (but are not enough to confirm) the idea that high-grade AAH is Eric Gilson closely related to BAC. Division of Environmental Medicine, National Defense Medical College Research Institute, Tokorozawa 359-8513, Japan [K. N.]; Department of Pathology, National Defense Medical College, INTRODUCTION Tokorozawa 359-8513, Japan [T. K., F. K., S. H.]; Division of AAH2 of the lung, a proliferation of alveolar epithelial Diagnostic Pathology, [M. M.] and Pathology [E. I.], Keio University cells, is usually found incidentally in lungs surgically resected School of Medicine, Tokyo 160-8582, Japan; and Laboratoire de for lung cancer, predominantly in patients with adenocarcinoma Biologie Mole´culaire et Cellulaire de l’Ecole Normale Supe´rieure de Lyon, UMR 5665 CNRS/ENSL, 69364 Lyon Cedex 07, France (1). In the 1999 WHO histological classification of lung and [C. E. K., E. G.] pleural tumors (2), AAH was classified among the preinvasive lesions along with squamous dysplasia, carcinoma in situ, and diffuse pulmonary neuroendocrine cell hyperplasia. However, ABSTRACT the relationship between AAH and pulmonary adenocarcinoma Purpose and Experimental Design: It has been suggested is not well understood. Recently, attempts have been made to that atypical adenomatous hyperplasia (AAH) may be a establish the pathogenesis of BAC by morphometric, immuno- precursor of peripheral adenocarcinoma of the lung. Telom- histochemical, and molecular studies (3, 4). The authors (3, 4) erase is a ribonucleoprotein enzyme that synthesizes telo- suggested that AAH should be regarded as representing a pre- meric DNA onto chromosomal ends. Its activity is thought to cancerous lesion or even an early-stage lesion of peripherally participate in the development of most human cancers. Te- located BAC. lomere-specific DNA-binding proteins, such as telomeric re- Telomerase is a ribonucleoprotein enzyme that synthesizes peat binding factor 1 and telomeric repeat binding factor 2, telomeric DNA at the end of chromosomes and compensates for also control telomere length in a complex interplay with the end replication problem, allowing cells to proliferate indef- telomerase. Here we investigated the expressions of the initely (5–7). Using the PCR-based telomerase repeat amplifi- mRNAs encoded by the TERF1 and TERF2 genes using in cation protocol assay, it has been shown that telomerase is situ hybridization in surgically resected specimens [28 AAHs activated in a large majority of human cancer tissues, but not in (11 lesions were interpreted as low-grade AAH, and 17 were most normal tissues or in tissues adjacent to malignant or benign interpreted as high-grade AAH) and 40 peripherally located tumors (8). Therefore, it appears that telomerase activity is a bronchioloalveolar carcinoma (BAC). useful marker for cancer detection. Results: A clear overexpression of these mRNAs was Two major subunits of the human telomerase core com- recognized in low- and high-grade AAH and BAC samples plex, namely, hTERC and hTERT, have been identified. (as compared with normal tissues) using in situ hybridiza- hTERC, which was identified first, functions as a template for tion and these mRNAs were detected in normal AAH and telomere elongation by telomerase (9–11). hTERT contains a BAC samples using reverse transcription-PCR. The expres- reverse transcriptase domain that catalyzes this reaction (12– sions of TERF1 and TERF2 mRNA detected by in situ 14). Recent studies have demonstrated that hTERC and hTERT hybridization were scored positive in 36% and 82% of form the minimum complex needed for telomerase activity (13, low-grade AAH, 65% and 83% of high-grade AAH, and 14). Therefore, up-regulation of hTERC and hTERT expres- 88% and 88% of BAC, respectively. Statistically significant sions could play an important role in human carcinogenesis. differences in TERF1 mRNA expression could be shown More recently, telomere-specific DNA-binding proteins such as between low-grade AAH and BAC and between high-grade TRF1 and TRF2 have been put forward as additional candidates AAH and BAC. There was no statistical difference in the for the role of molecules modifying telomerase activity, and they have been suggested to play key roles in the maintenance of telomere function (15–18). In fact, TRF1 negatively regulates Received 4/29/02; revised 10/29/02; accepted 11/4/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to 2 The abbreviations used are: AAH, atypical adenomatous hyperplasia; indicate this fact. TRF, telomeric repeat binding factor; BAC, bronchioloalveolar carci- 1 To whom requests for reprints should be addressed. Phone: 81-42- noma; RT-PCR, reverse transcription-PCR; hTERC, human telomerase 995-1505; Fax: 81-42-996-5192; E-mail: [email protected]. RNA component; hTERT, human telomerase reverse transcriptase. Downloaded from clincancerres.aacrjournals.org on September 23, 2021. © 2003 American Association for Cancer Research. 1106 TRF1 and TRF2 mRNAs in AAH the maintenance of telomere length (15, 16, 18). Although TRF2 was initially implicated in the protection of chromosome ends (15, 16), more recent reports show that TRF2 protein is a second negative regulator of telomere length (17) acting at least in part independently of telomerase by activating a degradation path- way (18). Although several investigations have been made of telom- erase activity and/or the subunits of human telomerase in lung carcinomas and AAH (11, 19–24), no studies have investigated the expressions of TERF1 and TERF2 mRNAs in AAH. We therefore examined their expressions in the AAHs and periph- erally located BACs (measuring 20 mm or less in the greatest diameter) that were available to us and compared the results with those obtained previously for hTERC and hTERT mRNA expressions in the same specimens. For this, we used primarily an in situ hybridization approach and also the RT-PCR. MATERIALS AND METHODS From 1980 to 2000, the surgical pathology service of the National Defense Medical College Hospital (Tokorozawa, Ja- pan) and Keio University Hospital (Tokyo, Japan) received specimens of 28 AAH lesions of the lung resected from 21 patients and 40 peripherally located BACs of the lung resected from 40 patients. In this study, we defined AAH lesions accord- ing to the following criteria: (a) AAH is composed of columnar or cuboidal cells arranged in a single row along the alveolar wall; (b) AAH cells are distinct from ciliated cells of the terminal bronchiolar epithelium; and (c) lung tissue surrounding Fig. 1 AAH. A, low-grade AAH; B, high-grade AAH. Scale bar, AAH does not exhibit a chronic inflammatory reaction in the 200 m. interstitial alveolar wall (Fig. 1). In situ hybridization was performed essentially as de- scribed previously (25). Briefly, sections were treated with 0.2 N HCl for 20 min and then incubated in 2ϫ SSC for 10 min at The corresponding sense probe was used for the negative con- 37°C and incubated in 5 g/ml proteinase K for 10 min at 37°C. trol. In situ hybridization of hTERC and hTERT mRNA was Sections were subsequently postfixed in 4% paraformaldehyde determined as described previously; the technique used and the for 5 min and then incubated in 0.1 M triethanolamine buffer (pH results in these same patients have been reported elsewhere (28). 8.0) containing 0.25% (v/v) acetic anhydride for 10 min to For analysis of reactivity, the extent of staining was scored as prevent nonspecific binding due to oxidation of the tissue. follows: (a) Ϫ, a negative reaction of tumor cells; (b) Ϯ, Յ10% Hybridization was carried out overnight at 42°C in 50% (v/v) of tumor area stained; (c) ϩ,11–25% of tumor area stained; (d) deionized formamide, 5ϫ Denhardt’s solution, 5% (w/v) dex- 2ϩ,26–50% of tumor area stained; and (e)3ϩ, Ն51% of tumor tran sulfate, 2ϫ SSC, 0.3 mg/ml salmon sperm DNA, 5 mM area stained. Those tumors in which the stained tumor cells EDTA, and 10 ng/ml biotin-labeled probes. After performing a made up Ͼ10% of the tumor were graded as positive. final stringent wash at 55°C for 20 min, hybridization was For examination of TERF1 and TERF2 mRNAs by RT- detected immunologically. PCR, total mRNAs were obtained from six normal lung tissues, pflagTRF1 (26) was digested by restriction enzymes EcoRI one high-grade AAH, and six BACs (the only suitable materials and XhoI, ligated between the EcoRI and XhoI cloning sites of available from our stock). The total RNA was isolated using pGEM7Zf (Promega), and then labeled with biotin using a RNA acid guanidinium isothiocyanate-phenol-chloroform extraction labeling kit (Boehringer Mannheim). The antisense probe was a and ethanol precipitation (29). RT-PCR was performed using an 247-bp segment of TERF1 cDNA under a SP6 promoter, and the amplification reagent kit (TaqMan EZRT-PCR kit; Applied corresponding sense probe was a 247-bp segment of TERF1 Biosystems, Alameda, CA) with TERF1, TERF2, and glyceral- cDNA under a T7 promoter. pBSKϩTRF2 (27) was digested by dehyde-3-phosphate dehydrogenase primers. Six primers were restriction enzymes PvuII and PstI, ligated between the PvuII synthesized using an automated DNA synthesizer. Sequence and PstI cloning sites of pSP72 (Promega), and then labeled information for all of the PCR primers used is listed in Table 1, with biotin using a RNA labeling kit (Boehringer Mannheim).