Main

Lung cancer represents the leading cause of cancer-related death in industrial countries and comprises about 20% small-cell lung carcinoma (SCLC) and 80% of non-small-cell lung carcinoma (NSCLC). According to the new WHO histological classification (Travis et al, 1999), non-small-cell lung carcinoma include with the common types squamous cell carcinoma (SCC) and adenocarcinoma (ADC), a recently described entity, the basaloid carcinoma (BC), as a variant of large cell carcinoma undergoing a particularly poor outcome (Brambilla et al, 1992). Surgical resection at early-stage disease represents the treatment of choice for NSCLC. However, survival rates remain low fostering identification of new prognostic factors and therapeutic target such as telomerase with the aim of deciphering new modes of adjuvant therapies.

Telomeres, which represent the end of the eukaryotic chromosomes, shorten at each cell division because of incomplete replication by DNA polymerase (Henderson, 1995). This results in telomere shortening leading to chromosome degradation or end fusion and cellular senescence acting as a ‘mitotic clock’ (Harley et al, 1990; Hastie et al, 1990; Allsopp et al, 1992). In germ line cells as well as in tumour cells, telomerase, a ribonucleoprotein complex composed of a reverse transcriptase catalytic subunit (hTERT) that copies a template region of RNA subunit (hTERC), can synthesise telomeric DNA, therefore, allowing cells to proliferate indefinitely (Counter et al, 1992; Nakamura and Cech, 1998; Holt and Shay 1999). While both hTERC and hTERT are required for telomerase activity, hTERC is expressed rather ubiquitously, whereas hTERT is the only limiting factor since its expression is confined to cells expressing telomerase activity (Meyerson et al, 1997; Nakamura et al, 1997; Kolquist et al, 1998).

Telomerase activity (TA) evaluated by a sensitive PCR-based telomere repeat amplification protocol (TRAP) assay has been widely reported in various malignancies such as liver, colorectal, brain, prostate and breast cancers as well as leukaemia (Bacchetti and Counter, 1995; Counter et al, 1995; Langford et al, 1995; Carey et al, 1998; Tahara et al, 1999; Kawakami et al, 2000). Regarding malignancies arising in the thorax, several studies have demonstrated a telomerase activity in SCLC and NSCLC carcinomas including NE tumours and adenocarcinomas and their precursor lesion (namely atypical alveolar hyperplasia) as well as in pulmonary sarcomas and mesotheliomas (Hiyama K et al, 1995; Ahrendt et al, 1997; Yashima et al, 1997; Gomez-Roman et al, 2000; Kumaki et al, 2001, 2002; Nakanishi et al, 2002). Almost all SCLC and the majority of NSCLC display a substantial telomerase activity in 62–96% of the cases (Hiyama K et al, 1995; Albanell et al, 1997; Gomez-Roman et al, 2000). Since a close relationship has been demonstrated between elevated telomerase activity and a poor prognosis in neuroblastoma and gastric carcinoma (Hiyama E et al, 1995a, 1995b), several reports have also suggested that high TA or high hTERT mRNA levels should be correlated with a poor survival in stage I NSCLC (Marchetti et al, 1999, 2002; Wang et al, 2002). High levels of telomerase have also been associated with tumour recurrence, histological type, grade (Marchetti et al, 1999, 2002; Kumaki et al, 2001) or smoking status (Travis et al, 1999).

We observed a lower expression of hTERT in stage I NSCLC but no influence of telomerase levels of expression on survival rate. This point remains controversial (Hara et al, 2001; Toomey et al, 2001) although several authors strongly support the unfavourable prognostic value of a high telomerase expression in stage I NSCLC (Marchetti et al, 1999, 2002; Wang et al, 2002). Controversies regarding telomerase levels in tumours reside in the variety of technical approaches. Most previous data are based on telomerase activity measured by TRAP assay or quantification of hTERT mRNA by RT–PCR requiring samples containing at least 5000 viable tumour cells and obtained freshly in RNAse-free conditions. Sample contamination by telomerase negative normal epithelial or stromal cells might explain TRAP negative assay, whereas positive activated lymphocytes might contribute to a positive TRAP assay in the absence of telomerase activity in tumour cells (Cunningham et al, 1998; **narianos et al, 2000). A requirement of 70–80% of cancer cells seems reasonable in order to compare telomerase levels to external standards provided by cell lines (Marchetti et al, 2002). Furthermore, the exactitude of any measurement of telomerase activity is challenged by intratumoral heterogeneity of hTERT expression (Kumaki et al, 2001; Paradis et al, 2001). We and others have experienced successful hTERT in situ hybridisation approaches, which evaluate the level of transcription of hTERT (Kolquist et al, 1998; Soria et al, 2001; Wang et al, 2002). However, this technique remains time and labour-consuming. Therefore, the most promising tool for an in situ evaluation of telomerase expression is now represented by immunohistochemical detection of hTERT. To date, studies using noncommercially available antibodies have shown the nuclear expression of hTERT in tumour cells of various type, as well as in progenitor cells and activated lymphocytes and no expression in normal somatic cells. Among different commercially available antibodies against hTERT, only the monoclonal 44F12 antibody gave us both a unique and specific band on Western blotting and a clear-cut nuclear staining only in tumour component and activated lymphocytes. Furthermore, we found a high concordance between semiquantitative approaches of hTERT expression evaluated by immunohistochemistry and Western blotting on a same sample set.

A good correlation has been demonstrated between TRAP and hTERT immunohistochemical detection (Tahara et al, 1999; Kawakami et al, 2000; Kumaki et al, 2001, 2002) in colorectal tumours, liver tissues, lung cancer and mesothelioma. Our immunohistochemical approach in the setting of lung cancer was confronted to the TRAP assay as well as to hTERT in situ hybridisation and standard Western blotting. Similar profiles of RTA levels and hTERT staining scores were observed in lung tumours, higher levels being noted in SCLC and BC than in SCC and ADC. However in five cases where protein was detected by immunohistochemistry, telomerase activity was absent, and in 10 other cases, levels of telomerase expression evaluated by TRAP assay and immunohistochemistry were discordant. Such discrepancies might be explained by dilution of tumoral positive cells in the sample or by post-transcriptional and post-translational regulations of the protein quantitatively and qualitatively. As an example, the level of phosphorylation is able to control both telomerase activity (Li et al, 1997; Kang et al, 1999) and cytoplasmic vs nuclear localisation of hTERT (Kharbanda et al, 2000; Liu et al, 2001; Kyo and Inoue, 2002).

Interestingly, we reported for the first time a nucleolar localization of the catalytic subunit hTERT, preferentially located onto nucleolar structures in 45% of SCC and 42% of ADCs in contrast with its diffuse nuclear localization in all SCLC and 74% of BC. We have considered this pattern of staining as specific as it was observed in a number of TRAP and Western blot positive tumours. Indeed, compelling evidence has been provided that the assembly of hTERT subunit and hTERC RNA via box H/ACA motif takes place into the nucleolus favouring the hTERC maturation and the stabilization of the telomerase protein complex (Mitchell et al, 1999). Nucleolar localisation of hTERT seems also to occur independently of hTERC binding, suggesting that this phenomenon could correspond to a sequestration of hTERT away from its telomeric targets (Etheridge et al, 2002). In addition, subnuclear distribution of hTERT may vary according to cell cycle stage or DNA damage. Thus in normal cells, telomerase is released to the nucleoplasm during the S phase where it can add telomeric sequences to replicating chromosomes. In contrast, in response to ionising radiation, telomerase is excluded from the nucleoplasm and accumulated into the nucleolus in order to limit its accessibility to nontelomeric ends and to prevent its association to inappropriate substrates during the repair of NA breaks (Wong et al, 2002). Conversely in SV 40 transfected cells, oncogenic transformation triggers the releasing of hTERT into the nucleoplasmic compartment increasing the telomeric sequence synthesis (Wong et al, 2002). Since we report here a shorter survival in stage I NSCLC exhibiting a nucleolar pattern of staining, several hypotheses concerning the signification and the prognostic implication of nucleolar hTERT confinement may be proposed. The nucleolar localisation in some ADC and SCC is consistent with a regulated compartmentalised type of hTERT accumulation process where telomere elongation remains separated from DNA repair process during the S phase, thus protecting DNA from genetic instability and inopportune crisis (Wang et al, 2002). In contrast, concomitant nucleolar and nuclear distribution observed in aggressive tumours such as SCLC and BC is suggestive of a strong and aberrant increase of telomerase activation in fast-growing tumours that have acquired a large enough number of genetic lesions to escape senescence and apoptosis.

As telomerase inhibitors may be mainly effective after multiple cell divisions leading to cell death, they may have their greatest impact in combination with cytotoxic chemotherapy in advanced-stage disease and in high-grade tumours, such as BC and SCLC as well as in adjuvant therapy to surgery in early-stage disease. Although nucleolar localization in early NSCLC deserves specific attention, further studies need to be performed to improve our knowledge about telomerase regulation through its subnuclear distribution and cell cycle dependency in order to clarify the fundamental basis of the prognostic influence of hTERT nucleolar localisation.