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[Cancer Research 66, 3584-3592, April 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

Telomerase- and Alternative Telomere Lengthening–Independent Telomere Stabilization in a Metastasis-Derived Human Non–Small Cell Lung Cancer Cell Line: Effect of Ectopic hTERT

Andreas Brachner1, Soleman Sasgary1, Christine Pirker1, Chantal Rodgarkia1, Mario Mikula1, Wolfgang Mikulits1, Helga Bergmeister2, Ulrike Setinek3, Matthias Wieser1, Suet-Feung Chin4, Carlos Caldas4, Michael Micksche1, Christa Cerni1,{dagger} and Walter Berger1

1 Department of Medicine I, Institute of Cancer Research, and 2 Institute of Biomedical Research, Medical University of Vienna; 3 Institute for Pathology and Bacteriology, Otto Wagner Hospital Baumgartner Höhe, Vienna, Austria and 4 Cancer Genomics Program, Department of Oncology, Hutchison/Medical Research Council Research Centre, University of Cambridge, Cambridge, United Kingdom

Requests for reprints: Walter Berger, Department of Medicine I, Institute of Cancer Research, Medical University Vienna, Borschkegasse 8a, A-1090 Wien, Austria. Phone: 43-1-4277-65245; Fax: 43-1-4277-9651; E-mail: walter.berger{at}meduniwien.ac.at.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the majority of human malignancies, maintenance of telomeres is achieved by reactivation of telomerase, whereas a smaller fraction uses an alternative telomere lengthening (ALT) mechanism. Here, we used 16 non–small cell lung cancer (NSCLC) cell lines to investigate telomere stabilization mechanisms and their effect on tumor aggressiveness. Three of 16 NSCLC cell lines (VL-9, SK-LU-1, and VL-7) lacked telomerase activity, correlating with significantly reduced tumorigenicity in vitro and in vivo. Of the three telomerase-negative cell lines, only SK-LU-1 displayed characteristics of an ALT mechanism (i.e., highly heterogeneous telomeres and ALT-associated promyelocytic leukemia bodies). VL-9 cells gained telomerase during in vitro propagation, indicating incomplete immortalization in vivo. In contrast, NSCLC metastasis-derived VL-7 cells remained telomerase and ALT negative up to high passage numbers and following transplantation in severe combined immunodeficient mice. Telomeres of VL-7 cells were homogenously short, and chromosomal instability (CIN) was comparable with most telomerase-positive cell lines. This indicates the presence of an efficient telomere stabilization mechanism different from telomerase and ALT in VL-7 cells. To test the effect of ectopic telomerase reverse transcriptase (hTERT) in these unique ALT- and telomerase-negative tumor backgrounds, hTERT was transfected into VL-7 cells. The activation of telomerase led to an excessively rapid gain of telomeric sequences resulting in very long (~14 kb), uniform telomeres. Additionally, hTERT expression induced a more aggressive growth behavior in vitro and in vivo without altering the level of CIN. These data provide further evidence for a direct oncogenic activity of hTERT not based on the inhibition of CIN. (Cancer Res 2006; 66(7): 3584-92)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
During the malignant progression of cancer cells, the maintenance of telomere length is a crucial prerequisite for immortalization (1). Whereas shortening of telomeres occurs in normal somatic cells and leads to irreversible cell cycle arrest and senescence (2, 3), cancer cells acquire the ability to keep constant or sufficient long telomeres. Usually, this state is achieved by reactivation of telomerase (4, 5) via the expression of telomerase reverse transcriptase (hTERT), the catalytic component of telomerase (6, 7). hTERT together with the RNA component hTERC represents the core enzyme complex (8). Generally, expression of hTERT is the limiting factor for telomerase activity (TA) in human cells and tissues (9). Accordingly, ectopic hTERT expression can lead to unlimited yet not tumorigenic growth (10–12). Besides the well-characterized function in the telomerase complex, hTERT was suggested to harbor telomerase-independent oncogenic functions (13, 14). Therefore, as part of the potent antitumor barrier in adult somatic tissues, hTERT expression is strictly regulated by diverse processes, including transcriptional and post-transcriptional mechanisms (15).

An alternative telomere lengthening (ALT) mechanism has been described for cancer cells lacking TA (4, 5, 16, 17). ALT is thought to be based on homologous recombination and copy switching of telomeric repeats (18). Thus, ALT cells are defined by an extreme heterogeneity of telomere lengths accompanied by distinct nuclear structures called ALT-associated promyelocytic leukemia (PML) bodies (APB; ref. 19). Although ALT provides a feasible mechanism for telomere length maintenance, it seems to induce enhanced chromosomal instability (CIN) due to the presence of some very short telomeres (20). In certain tumor entities, including sarcoma and glioblastoma multiforme, ALT is particularly common (21, 22). In case of glioblastoma, ALT correlated with a better patient prognosis, whereas no influence was detected for osteosarcoma. Interestingly, there exists a significant number of cancer cases exhibiting neither TA nor features of ALT (21, 23). Correspondingly, two research groups recently described a SV40-immortalized human cell line exhibiting neither TA nor APBs (24, 25). Furthermore, a reversible conversion of TA-positive to TA-negative cells has been reported in case of human papillomavirus type 16 E6-immortalized fibroblasts (26). This suggests the existence of additional effective mechanisms of telomere stabilization.

In a survey of telomere stabilization mechanisms in 16 human non–small cell lung cancer (NSCLC) cell lines, we found that, as expected, most investigated cancer cell lines were TA positive. Surprisingly, one squamous cell carcinoma–derived cell line (VL-7) lacked both TA and features associated with ALT, including APBs, heterogeneous telomere lengths, and enhanced CIN. These observations indicate the existence of a currently unknown, naturally occurring ALT mechanism conferring proper telomere stabilization and immortalization in VL-7 cells. When grown in severe combined immunodeficient (SCID) mice, VL-7 cells formed slow-growing tumors. Ectopic hTERT expression induced ultralong telomeres and aggressive tumor growth independent of changes in CIN. These findings provide direct evidence for a CIN-independent oncogenic function of hTERT.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and electroporation. The normal human embryonic lung fibroblast strain WI38, the osteosarcoma cell line U2-OS, and the human lung cancer cell lines A549, A427, Calu-6, and SK-LU-1 were obtained from American Type Culture Collection (Manassas, VA). The human lung cancer cell lines VL-1 to VL-10 were established from lung cancer patient material (27). All cell lines were grown in RPMI 1640, except WI38 and SK-LU-1 in MEME and U2-OS in Iscove's modified Dulbecco's medium at 37°C and 5% CO2 supplemented with 10% FCS and 100 units/mL penicillin/streptomycin. Cells were transfected with either pSP-hTERT or pSP as vector control by standard electroporation procedures (Equibio EasyjecT Plus). Stable clones were selected by exposure to 200 µg/mL G418 (Calbiochem, San Diego, CA). Minimal doubling times (MDT) were determined from growth curves as described (28).

Plasmids. To obtain pSP-hTERT expression vectors, the plasmid pGRN145 (kindly provided by Geron, Inc.) was cut with EcoRI and the purified insert was cloned into the EcoRI site of the pSP expression vector (29).

Telomeric repeat amplification protocol assay. The PCR-based telomeric repeat amplification protocol (TRAP) assay for detection of TA was done as described (30). The amplification products were separated on a 10% nondenaturing polyacrylamide gel, stained with Vistra Green (Amersham Biosciences, Aylesbury, United Kingdom) and visualized in a FluorImager 595 (Molecular Dynamics, Little Chalfont, United Kingdom). Semiquantitative densitometric evaluation was done by ImageQuant 5.0 (Molecular Dynamics).

Analysis of terminal restriction fragment length. Cells were lysed in proteinase K buffer [100 mmol/L NaCl, 10 mmol/L Tris (pH 8.0), 1 mmol/L EDTA, 0.7% SDS] and digested with 250 µg proteinase K at 50°C for 16 hours. Genomic DNA was extracted by phenol/chloroform method and precipitated with ethanol. DNA (10 µg) was digested with 30 units HinfI (Invitrogen, Carlsbad, CA) and 30 units RsaI (Invitrogen) in REAct 1 buffer for 16 hours at 37°C. For pulsed-field gel electrophoresis (PFGE), digested DNA samples were separated on a 1% pulsed field grade agarose gel (Bio-Rad, Hercules, CA) in 0.5x Tris-borate EDTA (TBE) for 7 hours at constant 9 V/cm using a pulse time of 0.1 second in a CHEF-DR III PFG apparatus (Bio-Rad) at 14°C. Following electrophoresis and ethidium bromide staining, gels were dried on filter papers for 1 hour at 60°C. Oligonucleotides (TTAGGG)4 were end-labeled with [{gamma}-32P]ATP and purified using NICK Columns Sephadex G-50 DNA grade (Amersham Biosciences). Gels were prehybridized for 4 hours at 37°C in 10 mmol/L Na2HPO4, 0.84 mmol/L Na4P2O7, 5x Denhardt's solution, and 4x SSC and hybridized with the probe for 24 hours at 37°C in 20 mL prehybridization solution. Then, gels were washed twice for 20 minutes in 0.1x SSC at 37°C. Image analysis was done using a PhosphorImager (Molecular Dynamics) and ImageQuant 5.0 for terminal restriction fragment (TRF) quantification.

Two-dimensional PFGE. Genomic DNA (30 µg) was digested with RsaI and HinfI and separated for the first dimension in a 0.5% agarose gel in 0.5x TBE with 2 V/cm and a pulse of 1 to 6 seconds for 12 hours on a CHEF-DR III PFG apparatus. The second dimension was done in a 1.1% agarose gel in 0.5x TBE with 4 V/cm and a pulse of 1 to 6 seconds for 8 hours. The gels from the first and second dimensions were stained with Vistra Green to confirm proper separation of the marker, a mixture of XhoI-digested {lambda}-DNA and a 1-kb marker (MBI Fermentas, Burlington, Canada). Gels were further processed and hybridized as for TRF analysis.

Reverse transcription-PCR. Reverse transcription-PCR (RT-PCR) was done as described (31) using the following oligonucleotide primers: hTERT: forward 5'-GCCTGAGCTGTACTTTGTCAA-3' and reverse 5'-CGCAAACAGCTTGTTCTCCATGTC-3', hTERC: forward 5'-TCTAACCCTAACTGAGAAGGGGCG-3' and reverse 5'-TTTGCTCTAGAATGAACGGTGGAAG-3', and glyceraldehyde-3-phosphate dehydrogenase: forward 5'-CGGGAAGCTTGTGATCAATGG-3' and reverse 5'-GGCAGTGATGGCATGGACTG-3'. PCR products were separated on a 5% nondenaturating polyacrylamide gel in a Protean-III electrophoresis chamber (Bio-Rad), stained with Vistra Green (Amersham Biosciences), visualized on a FluorImager 595, and adapted for print with ImageQuant 5.0.

Fluorescence in situ hybridization analysis. For quantitative fluorescence in situ hybridization (Q-FISH), metaphase spreads from lymphocytes of a healthy donor were prepared by standard methods. Tumor cell lines were seeded at low density and cultured in growth medium with 10% FCS for 24 hours. Colcemid (10 ng/mL) was given for 60 minutes and metaphase spreads were prepared according to standard methods. Slides were aged at room temperature for 1 week or at 90°C for 1 hour. Telomere length based on telomere signals of individual chromosomes was detected using the Telomere PNA FISH kit/FITC according to the manufacturer's instructions (DAKO, Carpinteria, CA). Metaphases were captured by Q-FISH software with a Leica DMRXA fluorescence microscope (Leica Mikroskopie und System, Wetzlar, Germany) equipped with appropriate epifluorescence filters and a COHU charged-coupled device camera. Images were analyzed by the Leica QWin software employing the image analysis and particle detection functions. Mean of fluorescence intensity of at least 400 individual telomeres was given in arbitrary units of telomere length. Variation of telomere length was calculated by the formula: SD / mean x 100.

For analysis of CIN, centromers of up to seven specific chromosomes were stained by FISH using centromeric probes to chromosomes 3, 7, 8, 12, 15, 17, and 18 (Qbiogene, Cambridge, United Kingdom and Vysis, Surrey, United Kingdom) in two-color FISH following the manufacturer's instructions and counted in >150 interphase nuclei per cell line. Modal numbers for each centromere were confirmed by analyzing at least 10 metaphase spreads per cell line prepared as described above. Levels of CIN were expressed as the mean percentage of nuclei with nonmodal centromere counts.

Immunofluorescence. APBs were detected by simultaneous staining of PML and TRF2 protein as described (32) using the antibodies PML (N-19) and TRF2 (H-300; both from Santa Cruz Biotechnology, Santa Cruz, CA) at dilutions of 1:100 and 1:200, respectively. As detection antibodies, FITC-conjugated anti-rabbit and Cy3-conjugated anti-goat antibodies (both from Sigma, St. Louis, MO) were used at dilutions of 1:500.

In vivo tumor growth. Cells of the desired cell type were detached from the tissue culture plate, washed with PBS, and resuspended in Ringer's solution (111 mmol/L NaCl, 1.9 mmol/L KCl, 1.1 mmol/L CaCl2, 2.4 mmol/L NaHCO3, 0.08 mmol/L NaH2PO4). Subsequently, aliquots of 1 x 106 cells in 100 µL Ringer's solution were s.c. injected into four SCID/BALB/c recipient mice. Tumor formation was measured periodically by palpation, and the tumor size was determined using a Vernier caliper. Tumor volume was calculated from tumor size using the formula: diameter x diameter x length / 2 as described previously (33). All experiments were done twice and carried out according to the Austrian guidelines for animal care and protection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Telomere and telomerase status of human lung cancer cell lines. A panel of 16 human NSCLC cell lines, mostly at early passage numbers, was analyzed with regard to TA and telomere status. Thirteen of 16 cell lines displayed TA at different levels, whereas 3 cell lines (SK-LU-1, VL-9 and VL-7) were TA negative. A representative TA analysis of 6 selected lung cancer cell lines compared with HeLa cells (positive control) and normal human embryonic lung fibroblast WI38 (negative control) is shown in Fig. 1A (left) . VL-9 cells, which were TA negative at low passage numbers, gained TA during in vitro cell culture (Fig. 1A, left, lanes 10 and 11). In contrast, neither SK-LU-1 nor VL-7 cells showed activation of telomerase up to high passage numbers (data not shown). To exclude the presence of any telomerase-inhibiting component in the TA-negative cell lines VL-7, VL-9 and SK-LU-1, HeLa lysates were added to the respective TRAP assays (Fig. 1A, right). No inhibition of HeLa TA was detected by any NSCLC cell extract tested. As negative control, lysates were preincubated with RNase.


Figure 1
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  		Figure 1. TA, telomerase component expression, and telomere lengths in NSCLC cell lines. A, TA was detected by TRAP assay in six selected NSCLC cell lines (lanes 2-7), telomerase-positive HeLa cells (lane 1), and telomerase-negative WI38 cells (lane 8). LB, lysis buffer only. Left, one of three independent experiments. Lanes 10 and 11, analysis of VL-9 cells at passage 21 (early) and passage 45 (late), respectively. Right, TA analysis of one TA-positive (VL-6) and three TA-negative (VL-7, VL-9, and SK-LU-1) NSCLC cell lines pretreated with RNase (lanes 2, 5, 8, and 11) or mixed with HeLa lysate (lanes 3, 6, 9, and 12), respectively. B, expression of the specific mRNA for hTERT (lane 1) and hTERC (lane 2) was analyzed by RT-PCR. The band at 457 bp corresponds to a functional hTERT full-length transcript (FL). The {alpha}-splice (421 bp), {alpha}+ß-splice (239 bp), and ß-splice variants (275 bp). HeLa and WI38 cells were included as TA-positive and TA-negative controls, respectively. Amplification of hTERC gave the expected 126-bp product. C, telomere lengths in NSCLC cell lines were determined by PFGE. HinfI- and RsaI-digested DNA, corresponding to 2 x 106 cells, was separated by PFGE and telomeres were visualized on hybridization with a telomeric probe (left). NSCLC cell lines were analyzed at early and late passages [population doubling level (PDL)]. Right, calculation of mean TRF size (in kb) by densitometric evaluation of the Southern blot. Open columns, mean TRFs at early PDLs; filled columns, mean TRFs at later PDLs.

 
To determine the underlying mechanism regulating TA, we did RT-PCR for the splice variants of the catalytic component of telomerase hTERT and the RNA component of telomerase hTERC (Fig. 1B). In SK-LU-1 and in the control WI38, no hTERT signal was detected. The other two TA-negative NSCLC cell lines (VL-7 and VL-9) only expressed the inactive hTERT ß-splice variant, generating a 275-bp PCR product. This indicated that at least the hTERT promoter and a portion of the coding regions were intact. As expected, the TA-positive cell lines, HeLa, VL-4, VL-6 and VL-8, expressed the functional full-length transcript of hTERT corresponding to a band at 457 bp. In addition, in these cell lines, the inactive {alpha}+ß- and ß-splice variants as well as minor amounts of the dominant-negative {alpha}-variant were detectable. As VL-9 cells exhibited low levels of TA in later passages (Fig. 1A, left), expression of hTERT splice variants at this time was analyzed. Indeed, full-length transcripts were weakly expressed by high-passage VL-9 cells (data not shown). With regard to expression of hTERC, all NSCLC cell lines were strongly positive without striking differences in expression levels (Fig. 1B).

Telomere lengths of NSCLC cell lines were determined at least at two time points during in vitro culture (Fig. 1C). The average TRF of four cell lines, surprisingly including the TA-negative VL-7 cells, resembled those of HeLa cells with stable telomere lengths ranging from 4 to 6 kb. Extremely short with a mean length of 2 kb, stable telomeres were detected in the TA-positive VL-8 cells. TA-negative SK-LU-1 cells contained very long telomeres with mean TRF of ~20 kb. Lack of TA together with the presence of long and variable telomeres are typical for cells displaying the ALT mechanism (17). Generally, telomeres of the investigated NSCLC cell lines remained fairly constant during in vitro culture irrespective of their TA status. However, TA-negative VL-9 cells lost telomere sequences during continuous culturing at an average of ~180 bp per cell generation. When telomeres became quite short, VL-9 cells spontaneously activated TA (Fig. 1A) and stabilized telomere lengths (data not shown).

Telomerase status compared with growth characteristics and CIN. The telomerase status of human NSCLC cell lines was significantly related to their in vitro growth potential (Fig. 2A ). The mean MDTs were significantly higher for the 3 TA-negative cell lines compared with the 11 TA-positive ones (38.2 ± 2.2 versus 29.8 ± 1.18 hours; P < 0.01, unpaired Student's t test). TA-positive NSCLC cell lines also exhibited increased tumor formation when implanted into the flanks of SCID mice (Fig. 2B). Whereas all 6 TA-positive cell lines formed tumors within 1 week, 2 of 3 TA-negative cell lines (VL-7 and VL-9) showed strongly delayed tumor formation and significantly slower tumor growth. SK-LU-1 cells did not form tumors in SCID mice.


Figure 2
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  		Figure 2. Growth potential and CIN of NSCLC cell lines compared with TA. A, MDTs for the investigated NSCLC cell lines were calculated from growth curves as described (28) and compared with the TA status obtained by TRAP assays. B, in vivo growth curves of the three indicated TA-negative (open symbols) and six representative TA-positive (closed symbols) NSCLC cell lines in the flanks of SCID mice. Points, mean tumor volumes of tumors grown in four SCID mice. C, for evaluation of CIN, centromers of seven different chromosomes were stained by FISH and counted in >150 interphase nuclei. Modal numbers for each centromere were confirmed by analyzing additionally at least 10 metaphases per cell line. Levels of CIN are expressed as the mean percentage of nuclei with nonmodal centromere counts and compared with regard to the TA status obtained by TRAP assays.

 
CIN was evaluated as far as possible at comparable passage numbers by counting centromers for seven different chromosomes in >150 cells per cell line (Fig. 2C). F2000 normal human embryonic fibroblasts were included as diploid controls to measure the percentage of false-positive detection of CIN. Generally, the number of cells with nonmodal chromosome numbers varied distinctly between cell lines. Levels of CIN did not differ statistically significant between TA-positive and TA-negative NSCLC cells. All three TA-negative cell lines had a comparable and intermediate level of CIN, whereas the TA-positive group contained very stable (VL-1 and A549) as well as excessively unstable cell lines (VL-10).

VL-7 cells do not exhibit features of ALT. The very long and variable TRFs detected in case of SK-LU-1 cells (compare Fig. 1C) are strongly indicative for the presence of an ALT mechanism of telomere stabilization (17). Surprisingly, this feature was missing in the second stably TA-negative NSCLC cell line VL-7. To further characterize the telomere maintenance mechanism of VL-7, the presence of APBs, another characteristic of ALT cells (32), was done and compared with the osteosarcoma cell line U2-OS, defined as ALT-positive (20). Whereas in SK-LU-1 (data not shown) and U2-OS, colocalization of PML protein and TRF2 in nuclear APBs was readily detectable, this feature was completely missing in case of VL-7 cells (Fig. 3A ). Another property of ALT-positive cells is the presence of extrachromosomal circular telomeric DNA (34), which can be visualized by two-dimensional PFGE (35). We detected circular telomeric DNA in the ALT-positive osteosarcoma cell line U2-OS but not in VL-7 cells (Fig. 3B). These results together with the TRF and TA analysis show that VL-7 cells lack both telomerase and ALT mechanisms for telomere stabilization.


Figure 3
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  		Figure 3. Markers of classic ALT in U2-OS and VL-7 cells. A, detection of APBs was done by immunofluorescence staining of PML protein (red) and the telomeric TRF2 protein (green) using nuclei (blue; 4',6-diamidino-2-phenylindole stain) of the indicated cell lines. The presence of APBs is indicated by a colocalization of the fluorescent signals (Merge) in U2-OS but not VL-7 cells. Bar, 10 µm. B, presence of extrachromosomal telomeric circles was analyzed by two-dimensional PFGE. Arrow, circular telomeric DNA detectable in U2-OS but not in VL-7 cells. Molecular weight in the first dimension is indicated in kb.

 
Effects of exogenous hTERT in TA- and ALT-negative VL-7 cells. hTERT expression in a telomerase-negative ALT background has been shown to induce TA and a tumorigenic phenotype (13). To investigate this phenomenon in a telomerase- and ALT-negative tumor cell background, VL-7 cells were transfected with hTERT or vector controls. Two VL-7 subclones (733 and 1012) showed high TA (Fig. 4A ), which was stably present during long-term in vitro culture. RT-PCR analysis (analogous to the experiments shown in Fig. 1B) indicated a correlation between the acquired TA and the expression of hTERT full-length transcripts in VL-7 subclones (data not shown).


Figure 4
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  		Figure 4. TA and telomere lengths in hTERT-transfected VL-7 subclones. A, TA of HeLa cells (positive control), parental VL-7 cells, and VL-7-derived subclones transfected with hTERT or the respective vector were analyzed by TRAP assays. A negative control containing lysis buffer only was included. TA during continuous propagation of TA-positive VL-7 subclones 733 and 1012 was analyzed at the indicated population doubling levels (lanes 8-11). B, telomere lengths in several VL-7 subclones determined by PFGE. The two TA-positive subclones (733 and 1012) harbor very long telomeres with a calculated TRF of 11.5 to 12.5 kb.

 
Telomere lengths in the various VL-7 subclones were determined at the earliest time point after transfection (Fig. 4B), which corresponded to ~20 cell generations between transfection, selection period, clonal isolation, and expansion. Whereas the TA-negative subclones had mean telomere lengths of 4 kb similar to the parental cells, telomeres of the TA-positive subclones 733 and 1012 exhibited mean TRFs of ~11 and 12 kb, respectively (Fig. 4B).

Besides TRF analysis by Southern blot, the telomere status of VL-7 clones was also investigated by Q-FISH analysis. The VL-7 vector control clones (1112 representatively in Fig. 5 ) as well as the parental VL-7 cells (data not shown) contained very short telomeres. In contrast, all chromatids of TA-positive clones (clone 733 in Fig. 5) exhibited very long and almost uniform telomeres. The homogeneity of telomere lengths was in sharp contrast to the broad variation of telomere lengths observed for ALT-positive SK-LU-1 cells (Fig. 5, bottom left). Lymphocytes from a normal donor displayed, as published (36), considerable telomere length variations, significantly lower as those observed for ALT-positive SK-LU-1 cells but distinctly higher as in the hTERT-expressing VL-7 subclone (Fig. 5, bottom right).


Figure 5
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  		Figure 5. Analysis of telomere lengths by Q-FISH. Metaphase spreads were prepared and telomeric repeats were detected by Q-FISH. Representative metaphases derived from VL-7 cell subclones transfected stably with hTERT (733) or the control vector (1112), the ALT-positive lung adenocarcinoma cell line SK-LU-1, and lymphocytes of a healthy donor. The lengths of at least 400 individual telomeres derived from at least three different metaphases were analyzed by quantifying fluorescence intensity of single telomeres using the QWin quantification software. Telomere lengths are mean arbitrary fluorescence units (left) and telomere variation was calculated as percent mean by the formula: SD / mean x 100 (right).

 
Processivity of exogenous hTERT in VL-7 cells. Dynamics of telomere elongation by exogenous hTERT was studied during continuous cell growth of TA-positive VL-7 clones 733 and 1012 (Fig. 6A ). hTERT expression induced extensive telomere lengthening with a distinct upper limit of ~14 kb. Moreover, assessment of the dynamics of telomere elongation revealed the addition of an average of 450 bp during each of the first 20 cell generations (i.e., after transfection) followed by a modest telomere elongation during the next 20 population doublings until the plateau level of 14 kb was reached.


Figure 6
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  		Figure 6. Dynamics of telomere elongation and growth characteristics and CIN of hTERT-transfected VL-7 subclones. A, TA-positive VL-7 subclones 733 and 1012 were continuously grown and changes in telomere lengths were analyzed by PFGE at different time points. Population doubling level with respect to the clonal founder cells in comparison with parental VL-7 and ALT-positive SK-LU-1 cells. Mean TRF for the TA-positive VL-7 subclones were calculated during increasing PDL (bottom). Mean TRF of parental VL-7 cells was taken as reference point. In vitro growth curves (B) and in vivo tumor formation (C) in SCID mice of VL-7 together with TA-positive and TA-negative subclones were determined. B, one of three comparable experiments. C, mean volumes of tumors grown s.c. in the flanks of four SCID mice. D, levels of CIN expressed as percentage of nonmodal cells with regard to the centromers of four chromosomes (3, 7, 12, and 17) were analyzed in VL-7 subclones with (hatched columns) and without (open columns) ectopic hTERT expression. Mean of >150 nuclei counted for each chromosome.

 
Effect of exogenous hTERT on growth characteristics as well as CIN in VL-7 cells. To investigate an effect of TA on VL-7 cell growth characteristics, hTERT-transfected and vector control VL-7 subclones were analyzed in vitro and in SCID mice. TA-positive VL-7 subclones gained a clear growth advantage compared with the TA-negative parental cells or vector control clones in vitro (Fig. 6B). This growth advantage was also detectable in vivo. A significantly enhanced s.c. tumor growth potential of the TA-positive clones compared with the parental cells and the hTERT-negative vector control clone was observed (Fig. 6C). Following growth in SCID mice, we aimed at reestablishing in vitro cell cultures. Whereas we succeeded in case of all TA-positive tumors, only one of five tumors in case of the vector control clones could be reestablished. TRF analyses of the reestablished cell clones showed preservation of the telomere lengths during in vivo tumor growth (data not shown).

To investigate whether the growth advantage of TA-positive VL-7 subclones is based on stabilization of chromosomes, levels of CIN were analyzed in TA-positive and vector control clones at comparable passage numbers. As shown in Fig. 6D, hTERT overexpression and consequent induction of long, uniform telomeres in VL-7 cells did not result in a significant change in the levels of CIN. This indicates that the growth-promoting activity of hTERT in a telomerase- and ALT-negative tumor background is not based on changes in chromosomal stability.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Most NSCLC especially at advanced stages exhibit TA, whereas ALT mechanisms of telomere stabilization seem to be rarely activated (16, 37, 38). Here, we investigated the association between telomere stabilization mechanisms and growth characteristics as well as genomic instability in a panel of 16 NSCLC cell lines. TA-negative NSCLC cell lines [3 of 16 (19%)] displayed a significantly less aggressive growth both in vitro and in SCID mice. The association between TA and growth behavior of NSCLC cells has not been investigated before this study. However, our in vitro and animal data corroborate several clinical investigations, all showing a significantly worse prognosis of lung cancer patients expressing TA and/or hTERT especially in stage I NSCLC (39–42). Furthermore, TA was found to correlate with enhanced clinical stage (37, 38). Concordantly, in all these studies, a subgroup of tumors was TA and/or hTERT negative even in advanced disease stages (37–42). This suggests that TA is not exclusively responsible for telomere stabilization and hence immortalization during development of NSCLC.

Thus far, one additional alternative mechanism for telomere maintenance, termed ALT, has been characterized (17). ALT is thought to involve recombinational events resulting in heterogeneous and ultralong telomeres resembling those found in yeast telomerase-negative type II survivors (43). Indeed, the TA-negative NSCLC cell line SK-LU-1 in our hands exhibited, in accordance with a previous observation (16), all features of ALT, including APBs (32) and the characteristic TRF length heterogeneity (17). ALT-positive tumors have been reported primarily from osteosarcoma and glioma patients (21, 22), whereas ALT-positive NSCLC specimens have only rarely been described thus far (16). Surprisingly, in two further TA- and hTERT mRNA-negative NSCLC cell lines, we could not detect characteristics of ALT. VL-9 cells, established from a stage II squamous cell carcinoma patient, underwent shortening of telomeres during in vitro culture followed by crisis, which led to outgrowth of TA-positive subclones. This points toward improper immortalization, and according to the telomerase hypothesis, VL-9 cells would represent mortal pre-M2-stage cells (44). In contrast, VL-7 cells established from a stage IV squamous cell carcinoma lymph node metastasis also lacked both TA and ALT but contained short but stable telomeres. Accordingly, these cells did not undergo crisis and did not reactivate TA up to high passage numbers. Although VL-7 cells were comparably slow-growing in vitro, they were able to form tumors in SCID mice without changing their TA and telomere status. These data suggest that, in addition to telomerase and ALT, human cells might harbor yet undefined mechanisms of telomere stabilization allowing immortalization and complete malignant transformation.

Although such mechanisms have not been described on a molecular basis thus far, clinical observations repeatedly suggested the existence of TA- and ALT-negative tumors. For example, only 34% of glioblastoma multiformes exhibited TA, whereas 47% lacked both TA and ALT (21). In case of Ewing's sarcoma, Ulaner et al. (45) reported that 30% of tumors lacked both telomere stabilization mechanisms. Even in osteosarcoma, shown to be prone to activate ALT during crisis, still 70% were TA- and ALT-negative, indicating a positive patient prognosis (46). The existence of telomerase- and ALT-negative tumors might be underestimated in vitro. For example, in case of >100 glioblastomas analyzed thus far, we found that exclusively telomerase-positive tumors allow successful establishment of stable cell lines in vitro.5 Complementary to the evidence from clinical studies, two research groups recently described a SV40-immortalized Werner-mutant human fibroblast cell line, AG11395, lacking TA and ALT (24, 25). It was shown, however, that this cell line harbors "tandem array" telomeres consisting of repeated units of integrated SV40 and telomere sequences. These telomere tandem structures are similar to those containing Y' elements observed in yeast type I survivors (47). Comparable telomere structures are unlikely to be present in VL-7 cells because very short and homogenous telomeres at all chromosome ends were detected by Q-FISH. Seger et al. (48) recently reported that telomerase activation is not a prerequisite for in vitro immortalization and transformation of human fibroblasts by oncogenes. However, these transformed cells, despite forming tumors in the nude mouse, lacked telomere stabilization, showed continuous erosion of telomeric repeats, and underwent crisis during longer in vitro propagation. All these features were absent in VL-7 cells. Very recently, a spontaneous conversion of human papillomavirus type 16 E6-immortalized fibroblasts from TA-positive to TA-negative cells based on hTERT promoter methylation has been reported (26). In this case, TA- and ALT-negative cells grew for >240 population doublings without any signs of TA or ALT activation. Furthermore, an immortalized human cell line derived from an ALT-positive WI38 clone, however, maintaining telomeres in the absence of key features of ALT and telomerase has been reported (49). These findings agree with our data regarding VL-7 cells, showing efficient stabilization of short telomeres without TA or ALT by yet undefined mechanisms.

In sharp contrast to ALT cells, VL-7 cells contained on all chromosome ends relatively short and uniform telomeres and lacked both APBs and extrachromosomal telomeric circles. In these respects, VL-7 cells resembled those NSCLC cell lines spontaneously exhibiting TA. However, ectopic overexpression of hTERT led to a highly efficient elongation of all telomeres until a distinct plateau level at 14 kb was reached. This implies that telomeres of VL-7 cells remain in a unique condition different from those in TA-positive but also ALT-positive cell lines causing high proficiency to be elongated by telomerase. Very different observations have been reported when hTERT is overexpressed in ALT cell lines. Telomerase elongated only the shortest telomeres; however, the heterogeneity of telomere lengths persisted (50). In contrast, fusion of ALT cells with TA-positive tumor cells led to inhibition of ALT in cell hybrids resulting in continuous shortening of telomeres until the lengths of TA-positive cell lines were reached. In VL-7 cells, however, TA led to the generation of telomere sequences at maximum efficiency, indicating that these cells do not contain repressors of telomerase as have been reported for several ALT cell lines (51).

The reason why VL-7 cells did not activate hTERT during malignant progression to metastasis, as typical for most late-stage NSCLC (37, 38), is currently unknown. With respect to hTERT mRNA, all three TA-negative NSCLC cell lines lacked full-length hTERT transcripts as also described previously for other ALT cell lines (52). Whereas the ALT-positive SK-LU-1 cells did not contain any hTERT mRNA variant, both VL-7 and VL-9 expressed the ß-splice variant, which is incapable of reconstituting an active enzyme (8). This indicates the presence of an active hTERT promoter also in VL-7 cells and suggests that alternative splicing plays a significant role in TA activation of NSCLC cells. Comparably, expression of alternatively spliced hTERT mRNA molecules has been very recently described for TA- and ALT-negative lung carcinoids (53). However, in contrast to our VL-7 cells, these carcinoid cells underwent crisis during in vitro culture, suggesting them as mortal pre-M2 cells comparable with VL-9 NSCLC cells. The role of the ß-splice variant-encoded hTERT protein remains enigmatic, and whether it might be engaged in the proposed "capping function" of telomerase (54) remains to be determined.

In the panel of NSCLC cell lines investigated, the lack of TA correlated with reduced growth and tumorigenicity independent if the cells exhibited characteristics of ALT. Moreover, ectopic induction of TA by expression of hTERT significantly enhanced tumor growth of VL-7 cells in vitro and in vivo. These observations suggest a direct role of telomerase in the aggressiveness of NSCLC cell growth. Generally, the favorable prognosis of patients with ALT-positive tumors has been attributed to an enhanced genomic instability based on the recombinational events involving very long and short telomeres (20, 21). However, we did not detect a significantly higher level of CIN in TA-negative cell lines. Both SK-LU-1 and VL-7 cells exhibited a median level of CIN, whereas TA-positive cell lines ranged from very stable to highly unstable. Moreover, ectopic expression of hTERT did not lead to a significant reduction of CIN within the investigated passage numbers. This does not exclude that during longer in vitro culture changed levels of CIN might become obvious. However, the enhanced growth potential of hTERT-positive clones was detectable immediately after transfection. This suggests that hTERT and/or TA directly promote aggressive growth of NSCLC cells. Accordingly, several reports provide evidence that hTERT, in additional to its role in telomere elongation, harbors functions that might contribute to the oncogenic phenotype in malignant cells. Thus, ALT could not substitute for hTERT in transformation of human fibroblasts in vitro (13). In our hands, hTERT transfection into ALT-positive SK-LU-1 cells led to an enhanced clonogenic potency (data not shown), suggesting that hTERT exerts its oncogenic functions detectable in VL-7 cells also in an ALT-positive background. Moreover, despite the long telomeres of mice, hTERT expression enhanced transformation of murine cells and hTERT transgenic mice were prone to develop tumors (55, 56). One hypothesis suggests that hTERT in addition to telomere elongation has a capping function preventing end-to-end fusion and illegitimate recombination events at short telomeres (54). However, this would imply a direct effect of hTERT on the level of CIN (54), which could not be observed. Together, these data point toward a telomere-independent oncogenic function of hTERT in NSCLC cells. Correspondingly, ectopic hTERT expression was shown to change the expression pattern of several growth-related genes in favor of enhanced cell growth (14). In agreement with these results, we observed in an initial analysis an up-regulation of extracellular signal-regulated kinase phosphorylation in hTERT-positive cell clones (data not shown).

In summary, we describe a fully immortalized and tumorigenic human cancer cell line derived from late-stage NSCLC, which lacks both known telomere stabilization mechanisms (i.e., TA and ALT). Ectopic expression of hTERT in this unique TA- and ALT-negative tumor background led to a more aggressive growth potential in vitro and in SCID mice. This effect of hTERT was independent of CIN and thus should not be dependent on telomere erosion. Together, our data suggest VL-7 cells as a useful tool for investigating, in addition to its unique telomere status, the molecular mechanisms underlying the oncogenic activities of hTERT.


   Acknowledgments
 
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 indicate this fact.


   Footnotes
 
{dagger} Deceased Back

Note: This work is dedicated to C. Cerni who died from cancer during the preparation of this article.

5 Manuscript in preparation. Back

Received 8/10/05. Revised 12/15/05. Accepted 1/25/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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