CORRESPONDENCE

Re: Effects of N-(4-Hydroxy-phenyl)retinamide on hTERT Expression in the Bronchial Epithelium of Cigarette Smokers

Rayleen Bowman, Belinda Clarke, Edwina Duhig, Jill Larsen, Kwun Fong

Affiliations of authors: R. Bowman, Department of Medicine, University of Queensland, Brisbane, Australia; B. Clarke, E. Duhig (Department of Pathology), J. Larsen (Thoracic Oncology Laboratory), K. Fong (Department of Thoracic Medicine), The Prince Charles Hospital, Brisbane.

Correspondence to: Rayleen Bowman, M.B.B.S., F.R.A.C.P., C Floor, Clinical Sciences Bldg., Royal Brisbane Hospital, Herston QLD 4029, Australia (e-mail: R.Bowman{at}medicine.uq.edu.au).

Soria et al. (1) report detecting human telomerase reverse transcriptase (hTERT) mRNA in endobronchial biopsies of smokers using in situ hybridization. They found positive hybridization signals in histologically normal bronchial epithelia and submucosal glands as well as in metaplastic and dysplastic epithelia. Recently, we also determined that hTERT is expressed in macroscopically normal bronchial epithelia of smokers. We used a real-time polymerase chain reaction (PCR) assay to study the expression of hTERT in 28 patients who had surgical resection for non-small-cell lung cancer. All patients gave informed written consent, and the study was approved by the ethics committee of The Prince Charles Hospital. The patients had a mean age (± standard deviation) of 64 ± 11 years, and all were current or former smokers with a mean cumulative tobacco exposure of 49 ± 29 pack-years. Fifteen patients had adenocarcinoma, nine had squamous carcinoma, two had adenosquamous carcinoma, one had a large-cell neuroendocrine carcinoma, and one had large-cell carcinoma, according to the World Health Organization classification of histologic tumor types (2). On the basis of International Union Against Cancer (UICC) pathologic staging categories (3), 12 cases were stage I, nine cases were stage II, and seven cases were stage III. We examined macroscopically normal peripheral lung tissue, bronchial mucosa scraped from uninvolved proximal bronchus, and tumor tissue from each patient. We found detectable hTERT expression in 10 (36%) of 28 bronchial epithelial scrapes, in 18 (64%) of 28 tumors, and in only one (4%) of 28 peripheral lung samples. Real-time quantitation demonstrated that the levels of hTERT expressed in bronchial epithelia, relative to those of constitutively expressed 18S ribosomal RNA, varied over a 1000-fold range but were comparable to those expressed in the corresponding tumors (Fig. 1Go). We therefore draw the same inference that Soria et al. (1) drew from their results, that is, deregulation of hTERT expression may be one of the earliest events in bronchial carcinogenesis. In addition, on the basis of the similarity in quantitative expression between bronchial epithelium and invasive carcinomas, we conclude that full derepression of hTERT has already taken place in the nonmalignant bronchial epithelium of a proportion of smokers. The expression of hTERT in bronchial epithelia of smokers and the lack of hTERT expression in the bronchial epithelia of nonsmokers [three reported by Soria et al. (1); one from our study (data not shown)] is consistent with the possibility that telomerase expression is associated with epithelial regenerative and proliferative activities that are induced in response to exposure to the cytotoxic components of cigarette smoke. However, Soria et al. (1) found no direct association between hTERT expression and proliferative status, as assessed by Ki-67 labeling. Rather than directly increasing the proliferative rate of bronchial epithelial cells, it is more likely that telomerase derepression confers on those cells the potential to replicate indefinitely. In a mutagenic environment, indefinite replicative potential removes a protective control by facilitating the fixation of accumulated mutational damage in replication. To further define the role of deregulated telomerase expression in bronchial carcinogenesis, we need to know whether telomerase expression is ever detectable in the epithelium of nonsmokers by studying larger numbers of nonsmokers. We also need to know whether telomerase is expressed in the epithelium of active smokers who have low cumulative tobacco exposure. Such individuals are likely to have changes in their bronchial epithelial injury and repair processes but are not likely to have a substantially increased risk of lung cancer.



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Fig. 1. Relative quantitation of hTERT expressed in the lung tumors ({blacksquare}) and the bronchial (Br) epithelium ({blacktriangleup}) of smokers. Tissues from fresh surgical specimens were snap-frozen in liquid nitrogen and stored at -70 °C. After homogenization in Trizol (Invitrogen, Groningen, The Netherlands), total RNA was extracted, and its quality was checked by gel electrophoresis. Ten microliters of total RNA was reverse transcribed by using random hexamers and Superscript II (Invitrogen) to produce complementary DNA (cDNA). Real-time polymerase chain reaction was performed by using an ABI PRISM 7700 sequence detector (Applied Biosystems, Foster City, CA) and the following amplification conditions: 95 °C for 10 minutes, followed by 40 cycles of 95 °C for 15 seconds and 60 °C for 1 minute. The hTERT probe and primers, which spanned the intron 13 splice site, were designed against the full-length hTERT cDNA sequence (Genbank accession No. AF015950) using Primer Express software (Applied Biosystems). A probe labeled with the VIC fluorophore (Applied Biosystems) and a set of oligonucleotide primers specific for 18S ribosomal RNA (Applied Biosystems) were used as the internal control to determine relative quantitation. Levels of hTERT mRNA are shown on the logarithmic y-axis as fold induction relative to expression of 18S ribosomal RNA in the same total RNA samples. Fold induction was calculated as described in the ABI Prism Sequence Detection System User Bulletin Number 2.

 

NOTES

Supported by The Prince Charles Hospital Research Foundation and the Queensland Cancer Fund.

REFERENCES

1 Soria JC, Moon C, Wang L, Hittelman WN, Jang SJ, Sun SY, et al. Effects of N-(4-hydroxyphenyl)retinamide on hTERT expression in the bronchial epithelium of cigarette smokers. J Natl Cancer Inst 2001;93:1257–63.[Abstract/Free Full Text]

2 World Health Organization. Histological typing of lung and pleural tumours. Geneva (Switzerland): World Health Organization; 1999.

3 Sobin L, Wittekind C. International Union Against Cancer TNM classification of malignant tumours. New York (NY): Wiley; 1997. p. 93–7.


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