REPORT

Effects of N-(4-Hydroxyphenyl)retinamide on hTERT Expression in the Bronchial Epithelium of Cigarette Smokers

Jean-Charles Soria, Chulso Moon, Luo Wang, Walter N. Hittelman, Se J. Jang, Shi-Young Sun, J. Jack Lee, Diane Liu, Jonathan M. Kurie, Rodolfo C. Morice, Jin S. Lee, Waun K. Hong, Li Mao

Affiliations of authors: J.-C. Soria, C. Moon, L. Wang, S. J. Jang, S.-Y. Sun, J. M. Kurie, J. S. Lee, W. K. Hong, L. Mao (Molecular Biology Laboratory, Department of Thoracic/Head and Neck Medical Oncology), W. N. Hittelman (Department of Experimental Therapeutics), J. J. Lee, D. Liu (Department of Biostatistics), R. C. Morice (Department of Thoracic and Cardiovascular Surgery), The University of Texas M. D. Anderson Cancer Center, Houston.

Correspondence to: Li Mao, M.D., Molecular Biology Laboratory, Department of Thoracic/Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Box 432, 1515 Holcombe Blvd., Houston, TX 77030 (e-mail: lmao{at}mdanderson.org).


    ABSTRACT
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Telomerase activation plays a critical role in tumorigenesis. To determine the role of telomerase in early lung carcinogenesis and as a potential biomarker in chemoprevention trials, we analyzed the expression of the human telomerase reverse transcriptase catalytic subunit (hTERT) in bronchial biopsy specimens from cigarette smokers who were enrolled in a randomized, double-blinded, placebo-controlled chemoprevention trial of N-(4-hydroxyphenyl)retinamide (4-HPR). Methods: We obtained biopsy specimens from six predetermined sites in the bronchial tree from the 57 participants, before treatment and 6 months after treatment with 4-HPR or placebo. We used in situ hybridization to examine hTERT messenger RNA (mRNA) expression in 266 pretreatment (baseline) and post-treatment site-paired biopsy specimens from 27 patients in the 4-HPR-treated group and from 30 patients in the placebo-treated group. All statistical tests were two-sided. Results: At baseline, 62.4% (95% confidence interval [CI] = 53.9% to 71%) of the biopsy specimens obtained from the group treated with 4-HPR and 65.2% (95% CI = 57.4% to 73.1%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. After 6 months, 45.6% (95% CI = 36.9% to 54.3%) of the biopsy specimens obtained from the 4-HPR-treated group and 68.1% (95% CI = 60.4% to 75.8%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. The reduction in hTERT expression observed between the two treatment groups over time was statistically significant (P = .01) when we used the biopsy site as the unit of analysis, but not when we used the individual as the unit of analysis (P = .37). Conclusions: Telomerase is frequently reactivated in the lungs of cigarette smokers. The modulation of hTERT expression in 4-HPR-treated smokers suggests that a novel molecular mechanism underlies the potential chemopreventive properties of 4-HPR. hTERT expression is a promising potential biomarker for risk assessment and for the evaluation of the efficacy of chemopreventive agents in lung carcinogenesis.



    INTRODUCTION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lung cancer is a major cause of mortality worldwide. In the United States alone in 2000, an estimated 164 100 new cases of lung cancer were diagnosed and an estimated 156 900 deaths from lung cancer occurred (1). Improving the survival rate for patients with this disease requires a better understanding of tumor biology and the subsequent development of novel therapeutic strategies. It is generally believed that lung cancer develops through a multistep process of genetic and epigenetic alterations that include the activation of oncogenes and the inactivation of tumor suppressor genes and that the cumulative effects of these changes lead to malignant transformation.

Telomerase is a ribonucleoprotein enzyme that extends chromosome ends that have been shortened during successive cycles of cell division (2). Telomerase is composed of an RNA component (hTERC), a catalytic protein subunit (hTERT), and other telomerase-associated proteins whose functions remain to be established (3). Telomerase is expressed in the vast majority of human malignant cell lines and tumors but not in the corresponding benign tissues (3). Studies of human tumors and human tumor cell lines (3,4) have shown that telomerase activation plays a critical role in tumorigenesis by sustaining cellular immortality. Telomerase activation occurs in most small-cell lung cancers and in up to 85% of non-small-cell lung cancers (NSCLCs) (5,6). Overexpression of hTERC, the RNA component of human telomerase, has been observed at a very early stage in the pathogenesis of NSCLC (7). The expression of hTERC, however, is not limited to the cells that harbor telomerase activity. By contrast, hTERT, the telomerase catalytic subunit gene, has a more restricted expression pattern, and its expression is more closely associated with telomerase activity (8,9). The recent development of in situ hybridization techniques that can reliably detect hTERT messenger RNA (mRNA) has made it possible to examine the expression of this critical telomerase component at the single-cell level (1016).

Chemoprevention—the use of specific natural or pharmacologic compounds to reverse, suppress, or prevent carcinogenic progression to invasive cancer—is an important strategy to decrease lung cancer incidence and mortality. Several studies have shown that one class of chemopreventive agents, the retinoids, are potent inhibitors of the multistep carcinogenic process in the airway epithelium (17). Furthermore, it has been shown that 13-cis-retinoic acid can increase the expression of retinoid acid receptor-{beta} in the lungs of heavy smokers, which suggests that retinoid-signaling pathways can be activated in the bronchial epithelium (18). One of these retinoids, N-(4-hydroxyphenyl)retinamide (4-HPR), has demonstrated chemopreventive activity in animal models of mammary, prostate, lung, and bladder cancers (19). We (20) have recently reported the results of a randomized, double-blinded, placebo-controlled trial using 4-HPR in the chemoprevention of squamous metaplasia and dysplasia of the bronchial epithelium. Although 4-HPR was not effective in reversing squamous metaplasia, we hypothesized that it might modulate genetic alterations that affect the expression of specific biomarkers in the bronchial epithelium of cigarette smokers. One such biomarker could be hTERT, the rate-limiting component of telomerase. We therefore evaluated the frequency of hTERT expression in 266 site-paired bronchial biopsy specimens collected from 57 cigarette smokers during the 4-HPR chemoprevention trial.


    MATERIALS AND METHODS
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 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Clinical Samples

All bronchial biopsy specimens were obtained from chronic or former cigarette smokers who had a smoking history of at least 20 pack-years and who were enrolled in a randomized, double-blinded, placebo-controlled chemoprevention trial of 4-HPR (20). All participants in the trial underwent a screening bronchoscopy and had baseline biopsy specimens taken at six predetermined sites in the bronchial tree. Participants whose biopsy specimens had evidence of dysplasia or had a metaplasia index value greater than 15% were randomly assigned to receive 4-HPR (200 mg per day administered orally) or placebo for 6 months (20). At the end of 6 months, the bronchoscopic examination was repeated, and biopsy specimens were collected at the same sites from which the baseline biopsy specimens were collected to evaluate the bronchial tree for evidence of change. The biopsy specimens were fixed in formalin and embedded in paraffin. Samples of normal lung tissue from three nonsmoking patients, as well as samples of an invasive bronchoalveolar carcinoma, were obtained from the Department of Thoracic and Cardiovascular Surgery at The University of Texas M. D. Anderson Cancer Center, Houston, and were similarly processed to serve as controls for the mRNA in situ hybridization procedure.

Cell Lines

The NSCLC cell lines NCI-H157, NCI-H292, NCI-H460, and SK-MES-1 used in this study were obtained from the American Type Culture Collection (Manassas, VA). These cell lines were grown in RPMI-1640 medium with 10% fetal bovine serum (Life Technologies Inc. [GIBCO BRL], Rockville, MD). The telomerase-negative alternative lengthening of telomeres (ALT) cell line KB319 was a gift from Drs. John Murnane and Laure Sabatier, Radiation Oncology Research Laboratory, University of California, San Francisco (21), and was cultured under the same conditions as those used for the NSCLC cell lines.

Telomeric Repeat Amplification Protocol Assay

The TRAP-eze telomerase detection kit (Intergen, Purchase, NY) was used to detect telomerase activity in the NSCLC and ALT cell lines according to the manufacturer's protocol, with minor modifications. For each cell line tested, 5 x 105 cells were lysed in microcentrifuge tubes containing 0.25 mL of cold lysate buffer (i.e., 0.5% 3-[(3-cholamidopropyldimethylamino]-1-propanesulfate, 10 mM Tris–HCl [pH 7.5], 1 mM MgCl2, 1 mM EGTA [i.e., ethylene glycol-bis(p-aminoethylether)], 10% glycerol, 5 mM {beta}-mercaptoethanol, and 0.1 mM benzamidine) for 30 minutes on ice. The lysates were centrifuged at 12 000g for 20 minutes at 4 °C, and the resulting supernatants were collected and stored at -80 °C. Heat-inactivated lysates were generated by incubating the supernatants at 85 °C for 10 minutes immediately before the telomeric repeat amplification protocol (TRAP) assay was performed.

The TRAP assay was performed in a 12.5-µL reaction mixture that contained 1 µL of supernatant (equivalent to 1000 cells), 50 mM deoxynucleoside triphosphates, 25 ng of telomerase substrate (TS) primer end-labeled with [{gamma}-32P]adenosine triphosphate, primer mix (RPTM primer, K1TM primer, and TSK1TM template), and 1 U of Taq DNA polymerase (Life Technologies Inc.). After a 20-minute incubation at 30 °C in the thermocycler block to allow telomerase-mediated extension of the TS primer, we performed 30 cycles of polymerase chain reaction (PCR) by incubating the reactions at 94 °C for 30 seconds, followed by incubation at 58 °C for 30 seconds. Five microliters of 3x loading dye (0.125% bromophenol blue, 0.125% xylene cyanol, and 15% glycerol in water) was added to 10 µL of each PCR reaction, and a 3-µL aliquot of that mixture was separated on a 7% polyacrylamide–urea–formamide gel. Non-heat-treated and heat-treated supernatants were loaded in alternating lanes of the gel. The control template, TSR8, was used as a standard for estimating the amount of TS primers with telomeric repeats extended by telomerase in a given reaction and was loaded on the last lanes of the gel. The gel was then exposed to film for autoradiography.

Riboprobe Generation and RNA in Situ Hybridization

We used a TOPO TA cloning® vector (pCR®II-TOPO; Invitrogen, Carlsbad, CA) that contained a 430-base-pair (bp) EcoRV–BamH1 fragment of the hTERT complementary DNA (cDNA) (a gift from Drs. Robert A. Weinberg and William C. Hahn, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge) (10) to generate a digoxigenin-labeled RNA probe (riboprobe) specific for the antisense strand of the hTERT cDNA. The plasmid was linearized with EcoRV and then transcribed in vitro with SP6 RNA polymerase (Promega Corp., Madison, WI) with the use of a digoxigenin–uridine triphosphate-labeling mixture (DIG RNA labeling kit; Roche Diagnostics Inc., Indianapolis, IN). The resulting digoxigenin-labeled RNA probe was mixed with ribonuclease (RNase) inhibitor (Roche Diagnostics Inc., Indianapolis, IN) and stored in aliquots at -80 °C.

For in situ hybridization of riboprobes to RNA, paraffin-embedded tissue sections (4 µm) were mounted onto silane-coated slides (Sigma Chemical Co., St. Louis, MO). The sections were deparaffinized in xylene and then rehydrated in gradually decreasing concentrations of ethanol. The sections were then treated with 2.5 µg/mL proteinase K (Roche Diagnostics Inc.) for 15 minutes at 37 °C, washed thrice with 1x phosphate-buffered saline (PBS), and post-fixed in 4% paraformaldehyde for 5 minutes at room temperature. The sections were acetylated in 0.25% acetic anhydride and 0.1 M triethanolamine for 10 minutes and then dehydrated in ethanol before hybridization. The sections were prehybridized by incubation in hybridization buffer (10% 20x sodium saline citrate [SSC; 20x SSC is 3 M sodium chloride and 0.3 M sodium citrate], 50% deionized formamide, 250 µg/mL predenatured salmon sperm DNA, 100 mg/mL dextran sulfate, 2% 100x Denhardt's solution [2% Ficoll 400, 2% polyvinylpyrrolidone, and 2% bovine serum albumin], 2% dithiothreitol, and 400 µg/mL yeast transfer RNA) for 1 hour at 42 °C. The sections were then incubated at 42 °C for 4 hours in fresh hybridization buffer that contained the riboprobe at 800 ng/mL. After hybridization, the sections were washed two times for 5 minutes in 2x SSC and then incubated in 2x SSC containing 0.05% Triton X-100 and 2% normal sheep serum (Sigma Chemical Co.) for 2 hours with agitation at room temperature. The sections were rinsed in Buffer 1 (0.1 M maleic acid and 0.15 M NaCl [pH 7.5]) for 5 minutes at room temperature and then incubated in Buffer 1 containing 2% normal sheep serum and 0.3% Triton X-100 for 30 minutes at room temperature. The slides were then incubated overnight at 4 °C with an alkaline phosphatase-conjugated anti-digoxigenin antibody (Roche Diagnostics Inc.) diluted 1 : 500 in Buffer 1 containing 1% normal sheep serum and 0.3% Triton X-100. The slides were rinsed twice in Buffer 1 and then briefly in Buffer 2 (100 mM Tris–HCl, 100 mM NaCl, and 50 mM MgCl2 [pH 9.5]). Alkaline phosphatase was detected with the use of 5-bromo-4-chloro-3-indolyl phosphate and nitro-blue tetrazolium chloride as chromogens. The slides were then rinsed in Buffer 3 (10 mM Tris–HCl and 1 mM EDTA [pH 8]) and mounted with Aqua-Mount medium (Fisher Scientific Co., Houston, TX).

We used several approaches to validate the results of in situ hybridization. Sections treated with RNase before hybridization with the riboprobe were used as negative controls for RNA hybridization. For each batch of sections analyzed, we also tested a telomerase-positive cell line (NCI-H460) and a telomerase-negative cell line (KB319) (21), which served as positive and negative controls, respectively, for hTERT mRNA expression. Both of these cell lines were processed for in situ hybridization in the same way as bronchial biopsy specimens (i.e., fixed in formalin and embedded in paraffin). An invasive bronchoalveolar carcinoma, for which expression of telomerase was confirmed by the TRAP assay, was used as an additional positive control for hTERT mRNA expression. We confirmed that the RNA in the biopsy sections was intact by performing in situ hybridization to detect retinoid X receptor-{alpha} mRNA, which is expressed constitutively in bronchial epithelium (20). Finally, we compared the results of hTERT in situ hybridization to detect hTERT mRNA in the lung cancer cell lines NCI-H460, NCI-H292, NCI-H157, and SK-MES-1 and in the ALT cell line KB319 with those obtained with the use of the TRAP assay to detect telomerase activity in the same samples.

A biopsy specimen was considered to be positive for hTERT expression if we observed a clear cytoplasmic hybridization signal. Biopsy specimens with no hybridization signal were counted as negative for hTERT expression. We did not grade the intensity of the hybridization signals obtained in this study.

Immunohistochemical Analysis of Ki-67

One 4-µm tissue section from each baseline biopsy specimen was immunocytochemically stained for Ki-67 expression to evaluate proliferative activity within the biopsy specimen. The tissue sections were deparaffinized in xylene, rehydrated through an alcohol series, and then immersed in 3% H2O2 in methanol for 15 minutes to block endogenous peroxidase activity. We accomplished antigen retrieval by placing the slides in citrate buffer (pH 6.0) and heating them in the microwave for two 4-minute periods. The slides were first incubated in 2% horse serum in PBS at 37 °C for 30 minutes and then incubated overnight at 4 °C in the same solution containing a 1 : 10 dilution of MIB 1, a mouse anti-Ki-67 antibody (Zymed, South San Francisco, CA). The slides were brought to room temperature for 15 minutes, washed once each in PBSD (0.1% Tween 20 in PBS) and PBS, and then incubated with biotinylated anti-mouse immunoglobulin G (Vector Laboratories Inc., Burlingame, CA) in 2% horse serum in PBS for 30 minutes at 37 °C. The slides were sequentially washed with PBSD and PBS, then processed with the ABC kit (Vector Laboratories Inc.) according to the manufacturer's recommendation, and reacted with 0.5 mg/mL diaminobenzidine and 0.6% hydrogen peroxide to detect the bound biotinylated secondary antibody. The slides were lightly counterstained in hematoxylin, washed in water, allowed to dry, and mounted in Eukit (Calibrated Instruments, Hawthorne, NY).

The fraction of Ki-67-positive cells was enumerated separately in the basal and parabasal epithelial layers of the bronchial biopsy specimens and was expressed as the percentage of cells with positive nuclear staining (Ki-67 labeling index).

Statistical Analysis

hTERT expression was determined at baseline and at 6 months on study with the use of the biopsy site and the participant as the units of analysis. When the participant was used as the unit of analysis, that individual was considered to be positive for hTERT expression when at least one of the six biopsy sites was positive for hTERT mRNA. The McNemar test was performed to determine the change in hTERT expression before and after treatment with 4-HPR. A method using the generalized estimating equations (GEEs) with binary outcome and logit link was applied to compare the change in hTERT expression over time between the placebo-treated and the 4-HPR-treated groups (22). The model considers the correlation structure that the biopsy sites are nested within a patient. All statistical analyses were performed with the use of SAS software (version 6.12) (SAS Institute, Inc., Cary, NC). All P values were determined by two-sided tests. P values less than or equal to .05 were considered to be statistically significant.


    RESULTS
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We used in situ hybridization to examine hTERT mRNA expression in bronchial biopsy specimens obtained from patients who were enrolled in a randomized chemoprevention trial for 4-HPR. Sufficient histologic specimens for evaluating hTERT expression were available for only 57 of the 70 patients who were initially evaluated in this chemoprevention trial. Of those 57 patients, 27 were in the 4-HPR-treated group and 30 were in the placebo-treated group. Patient characteristics are summarized in Table 1Go.


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Table 1. Patient characteristics and treatment groups*
 
We analyzed 266 pretreatment and post-treatment site-paired biopsy specimens from the 57 participants in the 4-HPR chemoprevention trial. Because of the unavailability of either pretreatment or post-treatment biopsy specimens at certain sites in some of the 57 participants, we could not analyze site-paired biopsy specimens from all six bronchial sites for each of the 57 participants. Using the hTERT riboprobe, we detected hTERT mRNA in bronchial epithelial cells in both metaplastic and dysplastic lesions, as well as in a substantial proportion of normal-looking tissue biopsy specimens (Fig. 1Go, A and B). It is interesting that, in a few cases, we also detected hTERT mRNA in submucosal glands (Fig. 1Go, B). In positive biopsy specimens, the hybridization signal was always cytoplasmic and was abrogated by RNase treatment of the sections before hybridization with the riboprobe, suggesting that the signal was related to the presence of hTERT mRNA. Biopsy specimens that were negative for hTERT mRNA (i.e., that had no hybridization signal) also contained metaplastic (Fig. 1Go, D) and normal-looking (Fig. 1Go, E) areas. We clearly detected hTERT mRNA in an invasive bronchoalveolar carcinoma that was used as a positive control for hTERT expression (Fig. 1Go, C), whereas the telomerase-negative immortal cell line KB319 consistently showed no appreciable hybridization signal (Fig. 1Go, F). The hybridization signal for hTERT mRNA in the bronchial epithelium of positive biopsy specimens ranged in intensity from low/moderate to strongly positive and was detectable in a substantial proportion of cells that were distributed either heterogeneously throughout the sections or in clusters (Fig. 1Go, A). Biopsy specimens with absent or faint hybridization signals were counted as negative for hTERT mRNA. We also analyzed normal bronchial epithelial samples from three nonsmoking patients and failed to detect any hTERT mRNA expression (data not shown).



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Fig. 1. Human telomerase reverse transcriptase catalytic subunit (hTERT) messenger RNA (mRNA) expression in the bronchial epithelium of smokers. hTERT mRNA expression in 266 site-paired bronchial biopsy specimens from 57 smokers enrolled in the 4-HPR chemoprevention trial was determined by in situ hybridization with the use of a specific riboprobe. The hTERT riboprobe stained bronchial epithelial cells in metaplastic lesions (A) and in some normal-looking biopsy specimens (B). hTERT-positive biopsy specimens expressed low to high levels of hTERT mRNA in a substantial proportion of cells that were either heterogeneously distributed throughout the section or clustered as shown by the arrow in panel A. Submucosal glands were also stained in some cases, as shown by the asterisk in panel B. Panel C shows clear staining of an invasive bronchoalveolar carcinoma, which was used as a positive control. Biopsy specimens that were negative for hTERT mRNA expression also included metaplastic (D) and normal-looking (E) areas. The telomerase-negative and immortal alternative lengthening of the telomeres (ALT) cell line KB319 consistently showed no appreciable staining (F).

 
We tested the correspondence between an in situ hybridization signal for hTERT mRNA and telomerase activity in human tissue culture cells because the very limited size of the bronchial biopsy specimens available for this study precluded the analysis of frozen samples necessary to measure telomerase activity. We obtained a clear and strong cytoplasmic in situ hybridization signal for hTERT mRNA in the NSCLC cell lines NCI-H157, NCI-H292, NCI-H460, and SK-MES-1 (Fig. 2Go, A–D); no hybridization signal was observed with the ALT cell line KB319 (Fig. 2Go, E). These same cell lines were tested for telomerase activity with the use of the TRAP assay. A characteristic 6-bp ladder, the signature of telomerase activity, was observed for the four NSCLC cell lines, whereas no ladder was observed for KB319 cells, which do not use telomerase as a mechanism to maintain their telomeres (Fig. 2Go, F). Overall, we found a very good correspondence between hTERT in situ hybridization, telomerase activity (TRAP assay), and reverse transcription–PCR for hTERT (data not shown), when comparing these techniques in the NSCLC cell lines NCI-H460, NCI -H292, NCI -H157, and SK-MES-1 and the ALT cell line KB319.



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Fig. 2. Comparison of in situ hybridization to detect human telomerase reverse transcriptase catalytic subunit (hTERT) messenger RNA and telomerase activity. The non-small-cell lung cancer cell lines NCI-H157 (A), NCI-H292 (B), NCI-H460 (C), and SK-MES-1 (D) and the alternative lengthening of the telomeres (ALT) cell line KB319 (E) were hybridized in situ with a riboprobe specific for hTERT. The same cell lines were assayed for telomerase activity with the use of the telomeric repeat amplification protocol assay (F). The characteristic 6-base-pair (bp) ladder diagnostic for telomerase activity was detected by autoradiography after the reaction mixtures were resolved by polyacrylamide gel electrophoresis. Heat-inactivated samples served as negative controls for telomerase activity (F, lanes 2, 4, 6, 8, and 10). TSR8 served as a quantitation control for estimating the amount of telomerase substrate primers with telomeric repeats extended by telomerase and was loaded in the last two lanes of the polyacrylamide gel (F, lanes 11 and 12). The 36-bp internal control was present in all samples tested (arrow in panel F).

 
hTERT expression did not directly correspond to the presence of specific histopathologic features within the biopsy specimens. For example, we found that a positive hybridization signal for hTERT mRNA was not related to the presence of squamous metaplasia. Indeed, 73 (68.2%; 95% confidence interval [CI] = 59.4% to 77%) of 107 biopsy sites with evidence of squamous metaplasia expressed hTERT at baseline, compared with 93 (60.3%; 95% CI = 52.7% to 68.1%) of 154 biopsy sites without squamous metaplasia (P = .19). At baseline, the percentage of biopsy specimens that expressed hTERT mRNA in the 4-HPR-treated group (62.4%; 95% CI = 53.9% to 71%) was similar to that in the placebo-treated group (65.2%; 95% CI = 57.4% to 73.1%) (Table 2Go). However, after 6 months on study, only 45.6% (95% CI = 36.9% to 54.3%) of the site-paired biopsy specimens from the 4-HPR-treated group expressed hTERT mRNA, while 68.1% (95% CI = 60.4% to 75.8%) of the site-paired biopsy specimens from the placebo-treated group did. The reduction in the percentage of biopsy specimens expressing hTERT mRNA in the 4-HPR-treated group was statistically significant (P = .01, McNemar test). This modulation remained statistically significant when compared with changes in the percentage of biopsy specimens expressing hTERT observed in the placebo-treated group (P = .027, GEE method). When we used the individual as the unit for analysis, we found that 25 (83.3%; 95% CI = 70% to 96.7%) of 30 patients in the placebo-treated group had positive biopsy specimens at baseline as well as after 6 months, whereas 22 (81.5%; 95% CI = 66.8% to 96.1%) of 27 patients in the 4-HPR-treated group had positive biopsy specimens at baseline but only 19 (70.4%; 95% CI = 53.1% to 87.6%) of those 27 patients had positive biopsy specimens after 6 months of treatment. This trend of reduced hTERT mRNA expression after 6 months of 4-HPR treatment was not statistically significant (P = .37, McNemar test).


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Table 2. Human telomerase reverse transcriptase catalytic subunit (hTERT) expression by biopsy site and participant*
 
Because the relationship between hTERT expression and cell proliferation remains controversial in the literature, we compared the hTERT mRNA status with the expression of Ki-67, a marker of cell proliferation, for each of the baseline biopsy specimens. The proliferative status of the baseline biopsy specimens was assessed with the use of an antibody to Ki-67, and Ki-67 labeling indices were determined separately for the basal and para-basal layers of the bronchial epithelia, as was reported recently (23). We found no statistically significant difference between the Ki-67 para-basal labeling index of hTERT-positive biopsy specimens (median labeling index = 0.1) and that of hTERT-negative bronchial biopsy specimens (median labeling index = 0.094; P = .68, Wilcoxon test) or between the Ki-67 basal labeling index of the hTERT-positive biopsy specimens (median labeling index = 0.048) and that of the hTERT-negative biopsy specimens (median labeling index = 0.053; P = .76, Wilcoxon test) (data not shown).


    DISCUSSION
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The recent randomized trial of 4-HPR in the chemoprevention of squamous metaplasia and dysplasia of the bronchial epithelium provides a unique opportunity to systematically measure hTERT mRNA expression in carcinogen-damaged lungs that have no clinically detectable cancer. In this study, we found that hTERT mRNA was detectable in about 64% of the bronchial biopsy specimens obtained from participants in the trial at baseline. Therefore, to our knowledge, this study is the first to demonstrate that hTERT mRNA is expressed frequently in bronchial epithelial cells obtained from chronic smokers without lung cancer. Given the close correlation between hTERT mRNA expression and telomerase activity that has been demonstrated in many studies (810), our observation suggests that telomerase is reactivated in these hTERT mRNA-expressing cells. This is consistent with results from a previous study, which found that telomerase reactivation and/or dysregulated telomerase RNA expression frequently occurred in bronchial lesions with carcinoma in situ, thus implicating telomerase in early lung carcinogenesis (7). Our results are also consistent with those of a recent report (24) that suggested that NSCLCs in smokers have higher telomerase activity than NSCLCs in nonsmokers.

We were able to detect hTERT mRNA in multiple biopsy sites in the bronchial tree from the same individual. In addition, we observed many areas of clustered hTERT expression within a biopsy specimen (Fig. 1Go, A). These findings suggest that multiple abnormal clonal lesions are present in carcinogen-damaged lungs and are consistent with our previous observation that smoking-damaged lungs contain widespread independent clones with genetic alterations that, among other things, can include cells that have reactivated telomerase (25). Of particular interest is the finding that hTERT expression was observed not only in biopsy specimens that displayed morphologic abnormalities, such as dysplasia or squamous metaplasia, but also in normal-appearing bronchial epithelium. hTERT positivity in normal-looking areas highlights the phenotypic and genotypic disparities that can exist in the bronchial epithelium.

The most important finding in our study was that 4-HPR treatment reduced hTERT mRNA in smoking-damaged lungs. Given the close correlation between hTERT mRNA expression and telomerase activity, this finding suggests that 4-HPR may also reduce telomerase activity in lungs damaged by smoking. At baseline, the rate of hTERT expression in the 4-HPR-treated group (62.4%) was similar to that in the placebo-treated group (65.2%). This finding indicates that expression of this biomarker was well balanced between both arms of the chemoprevention study. This consistency between the two treatment groups also indicates that the in situ hybridization technique used to detect hTERT mRNA is reliable. When we used the biopsy site as the unit of analysis, the reduction in hTERT expression in the 4-HPR-treated group after 6 months in the study was statistically significant (P = .01), and this modulation remained statistically significant when compared with changes observed in the placebo-treated group during the same time period (P = .027). However when the individual was considered as the unit of analysis, the trend of reduced hTERT mRNA expression after 6 months of 4-HPR treatment was not statistically significant. This lack of significance is probably related to the small sample size of patients tested (n = 27 in the 4-HPR-treated group).

We have considered two potential limitations to our study. First, because Albanell et al. (6) have shown that telomerase expression in patients with NSCLC can be associated with cell proliferation, one major concern was that the reduction in hTERT expression in the 4-HPR arm might have been caused by the effect of 4-HPR on cell turnover. To address this issue, we compared the expression of hTERT with that of Ki-67, a marker of cell proliferation, for each biopsy site at baseline. We found no statistically significant differences in Ki-67 labeling between the hTERT-positive and the hTERT-negative biopsy specimens. The lack of an association between the hTERT expression and the Ki-67 labeling index suggests that hTERT expression is not a simple marker of cell proliferation and that the observed effect of 4-HPR on hTERT mRNA expression is not directly related to the drug's effects on cell turnover.

Second, recent reports (26,27) have shown that most cancer samples contain the full-length transcript encoding functional hTERT as well as alternative transcripts that encode abbreviated and, in some cases, nonfunctional versions of hTERT. We have confirmed that alternative hTERT transcripts coexist with the full-length transcript in NSCLC cell lines as well as in a sample of invasive lung cancers (data not shown). Because the riboprobe used in our studies can hybridize to the full-length hTERT transcript and theoretically, through partial hybridization, to some of the alternative transcripts of hTERT, this raises the possibility that at least some of the in situ hybridization signal that we observe in the biopsy specimens is due to alternative hTERT transcripts that do not make functional telomerase. Nevertheless, we believe that the main conclusions of our study remain intact, even though the signal from our riboprobe may be related to both the full-length transcript and alternatively spliced transcripts. Indeed, we have rated the bronchial biopsy specimens as positive or negative for hTERT without considering the intensity of the hybridization signal. Thus, a positive signal is, at least in part, related to the full-length transcript, whereas a completely negative biopsy specimen reflects the absence of all hTERT transcripts. Because we have shown statistically significant qualitative changes in hTERT expression in the bronchial biopsy specimens after 4-HPR treatment, these results remain accurate, even with the signals that could be related to the alternatively spliced hTERT mRNAs.

The potential of 4-HPR to modulate hTERT expression gives insight into a new molecular mechanism through which this chemopreventive agent works. The mechanism by which 4-HPR mediates its chemopreventive effect has been investigated extensively. 4-HPR treatment of cancer cells in vitro activates the transcription of genes containing the retinoid acid response elements through retinoid nuclear receptor-dependent pathways and increases the expression of retinoic acid receptor-{beta} (28). In addition, 4-HPR induces apoptosis in a variety of cancer cell lines through both retinoid receptor-dependent and -independent mechanisms (29,30). Using in vitro systems, other investigators (3134) have demonstrated that retinoic acid induces cell differentiation that results in a pronounced reduction of telomerase activity. At low concentrations, 4-HPR induces cell differentiation rather than apoptosis though a retinoid receptor-dependent pathway (30). It is interesting that a recent report (35) has shown that 9-cis-retinoic acid and 13-cis-retinoic acid can prevent the immortalization of breast epithelial cells. A recent study (36) has also shown that 4-HPR suppresses cell proliferation and telomerase activity in an in vivo model of rat mammary tumors. However, it is not yet clear whether 4-HPR reduces expression of hTERT by apoptosis, differentiation, or some other mechanism. The recent identification of an estrogen-responsive element in the hTERT promoter has suggested the possibility that 4-HPR may directly influence hTERT mRNA levels in cells (37).

Although hTERT expression was reduced in the bronchial epithelium of smokers who received 4-HPR, this drug did not reverse pathologic changes in the bronchial mucosae of patients in the same cohort (20). One possible explanation for this result is that the dose of 4-HPR used in the chemoprevention trial was too low to trigger the cell death that was observed in in vitro experiments that used this agent at higher doses (38). Higher doses of 4-HPR are under active investigation in phase I studies. Nevertheless, these findings demonstrate that molecular markers, such as hTERT, are powerful tools to assess the efficacy of chemopreventive agents in clinical trials and may provide a rationale for conducting a trial using a higher dose of 4-HPR.

In conclusion, we have shown that hTERT expression is a frequent event and appears at a very early stage in cigarette smoking-induced lung carcinogenesis. We provide evidence to suggest that 4-HPR reduces hTERT expression in the bronchial epithelium of current and former smokers. Our results suggest that hTERT is a sensitive and promising biomarker to evaluate the efficacy of chemopreventive agents in the lung.


    NOTES
 
J.-C. Soria and C. Moon contributed equally to this work.

Supported by American Cancer Society grant RPG-98–054 (to L. Mao); by Public Health Service grants P01CA74173 and U19CA68437 (to W. K. Hong) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services; by the Fondation de France, Assistance Publique–Hôpitaun de Paris, and Lilly Foundation grant (to J.-C. Soria); and by the Tobacco Research Fund from the state of Texas (to The University of Texas M. D. Anderson Cancer Center, Houston). W. K. Hong is an American Cancer Society Clinical Research Professor.

We thank Dr. John Murnane and Dr. Laure Sabatier (Radiation Oncology Research Laboratory, University of California, San Francisco) for the telomerase-negative cell line KB319. We are indebted to Dr. Robert A. Weinberg and Dr. William C. Hahn (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA) for the full-length hTERT complementary DNA. We also thank Kate Ó. Súilleabháin for editing the manuscript and Sandra Ideker for the artwork.


    REFERENCES
 Top
 Notes
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Manuscript received December 15, 2000; revised May 31, 2001; accepted June 18, 2001.


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