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Carcinogenesis Advance Access originally published online on September 14, 2006
Carcinogenesis 2007 28(3):553-559; doi:10.1093/carcin/bgl158
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inhibition of gene amplification in telomerase deficient immortalized mouse embryonic fibroblasts

Paola Rebuzzini, Paola Martinelli, Maria Blasco1, Elena Giulotto2 and Chiara Mondello*

Istituto di Genetica Molecolare, CNR Via Abbiategrasso 207, 27100 Pavia, Italy
1 Spanish National Cancer Center, Melchor Fernández Almagro 3 28029 Madrid, Spain
2 Dipartimento di Genetica e Microbiologia ‘Adriano Buzzati Traverso’, University of Pavia Via Ferrata 1, 27100 Pavia, Italy

*To whom correspondence should be addressed. Tel: +39 0382 546332; Fax: +39 0382 422286; Email: mondello{at}igm.cnr.it


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mutations in genes important for the preservation of genome stability can increase the frequency of gene amplification, a process relevant to tumor development. To investigate whether telomerase, the enzyme deputed to telomere maintenance, also plays a role in gene amplification, we studied the amplification of the carbamyl-P-synthetase, aspartate transcarbamilase, dihydro-orotase (CAD) gene in immortalized embryonic fibroblasts derived from telomerase knockout mice (mTERC–/–) of the first and of the sixth generation. As expected, in 9 out of 10 N-(phosphonacetyl)-L-aspartate (PALA) resistant clones derived from wild-type cells, CAD was amplified; in contrast, in none of the 30 PALA resistant clones isolated from the three mTERC–/– cell lines we could detect CAD amplification, indicating that, in the absence of telomerase activity, gene amplification is inhibited. The causal relationship between mTERC deficiency and lack of gene amplification was demonstrated by the restoration of CAD gene amplification in two of the three deficient cell lines transfected with mTERC. The lack of amplification in mTERC deficient cells could be related to a defect in the stabilization of the ends of the amplified chromosomes in the absence of telomerase, to a more general effect of telomerase in the regulation of gene expression, including genes involved in amplification, or to a possible interaction of the telomerase RNA with proteins involved in gene amplification.

Abbreviations: PALA, N-(phosphonacetyl)-L-aspartate; BFB, breakage-fusion-bridge; TRF, terminal restriction fragment


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Gene amplification is a chromosomal rearrangement distinctive of cancer cells, consisting of an increase in the copy number of a region of the genome. In tumor cells, gene amplification plays an important role in the activation of proto-oncogenes, and thus in tumor progression (1).

In normal somatic cells, gene amplification has never been detected, suggesting that these cells are endowed with mechanisms that prevent the occurrence of gene amplification, or are committed to death when they bear amplified DNA (2,3). The lack of gene amplification in normal cells could be related to their tight control on the maintenance of genome integrity, which is, in contrast, loose in cancer cells. In this regard, it has been shown that the loss of p53 function is sufficient to make normal cells permissive for gene amplification (4,5). Gene amplification is influenced by the cellular genetic constitution, as shown by the isolation of hamster cells with a high propensity to amplify, called ‘amplificator’ mutants (6).

The amplified DNA can be located on extra chromosomal elements, devoid of the centromere but autonomously replicating, or within chromosomes. In this latter case, the presence of a high number of copies can give rise to abnormally banded chromosome regions [reviewed in (7)]. Several mechanisms can contribute to the genesis of amplified structures, mainly: aberrant replication of a genomic region, which can be followed either by the excision or by the integration of the over-replicated fragment, leading to either extra- or intrachromosomal amplification (8,9), unequal sister chromatid exchange (10) and breakage-fusion-bridge (BFB) cycles (11).

All these mechanisms are initiated by DNA breakage. In particular, BFB cycles can be triggered by DNA double-strand breaks (12–18). After DNA replication, if the break is not properly repaired, telomere-less sister chromatids are generated, which are prone to fuse, giving rise to dicentric chromosomes. Dicentrics can enter successive BFB cycles, until broken ends are stabilized by the addition of a telomere (7,19). We have recently shown that defects in the cellular response to DNA double-strand breaks increase the frequency of gene amplification, indicating that un-faithfully repaired DNA double-strand breaks can undergo promiscuous recombination and trigger gene amplification (20,21).

Evidence has also been reported that sister chromatid fusions can be driven by the presence of dysfunctional telomeres or by telomere loss (22,23).

Telomeres are the physical ends of eukaryotic chromosomes and are critical elements for chromosome stability (24). Telomeres protect chromosome extremities from nucleolytic attack, allow cells to distinguish between chromosome ends and broken sites, and prevent recombination between chromosomes. In vertebrates, telomeres are formed by tandem repetitions of the hexanucleotide TTAGGG bound to specific proteins (25). The length of the telomeric DNA tract is species-specific; in humans it ranges from 10 to 20 kb, depending on tissue and donor age, in mice it can extend up to 50 kb. Telomeres are maintained by telomerase, a reverse transcriptase whose main components are a catalytic subunit (TERT) and an RNA moiety (TERC), which contains the template for the synthesis of the telomeric repeats.

In the absence of telomerase, telomeres shorten at each cell division, because conventional DNA polymerases cannot replicate the 3' end of linear DNA molecules (26). When telomeres reach a critical length, they cannot fulfil their functions anymore, leading to chromosomal instability (27). A mouse model has been created in which the telomerase RNA gene has been ablated by homologous recombination (28). The knockout mice (mTERC–/–) are viable only for a limited number of generations; by the sixth generation, animals die because of telomere exhaustion. In late generation mice, telomere-less chromosomes are detected, together with a great number of end-to-end fusions and rearranged chromosomes, confirming the essential role of telomeres in the maintenance of genome stability (29).

In the work presented in this study, we aimed at testing whether telomerase plays also a role in gene amplification. For this purpose, we analyzed CAD gene amplification in immortalized telomerase deficient fibroblasts derived from mice of the first and the sixth generation (G1 and G6 mice, respectively) and in the same cell lines after restoration of telomerase activity.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Cells and cell culture
MEFs obtained from wild-type (WT17, PDs 78–88), first generation (KO16, PDs 85–95) and sixth generation (KO9F6, PDs 73–84; KO11F6, PDs 50–60) mTERC–/– mice had been immortalized in vitro with the 3T3 protocol (30,31). All the cell lines were grown in DMEM (Hy-Clone) supplemented with 10% FBS (Bio-Whittaker) and maintained at 37°C in the presence of 5% CO2.

N-(phosphonacetyl)-L-aspartate (PALA) was a kind gift of the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute (Bethesda, MD). Experiments with PALA were carried out in the presence of 1 µM dipyridamole (Boehringer Ingelheim), a specific inhibitor of the uptake of uridine, which increases PALA toxicity (32).

Measurement of sensitivity to PALA
PALA sensitivity was determined by measuring the inhibition of growth in massive cell culture. Cells (105 cells per 6 cm dish) were plated in medium containing increasing concentration of PALA and 1 µM dipyridamole. After 3 days, the cells were collected and washed with phosphate-buffered saline (PBS); then, cells were lysed in NaOH 0.1 M at 50°C for 30 min. The number of cells was estimated by measuring the absorbance of the lysed samples and, for each cell line, the dose of PALA reducing growth of 50% (RD50) was determined and was found to be ~20 µM PALA for wild-type cells and ~10 µM PALA for telomerase deficient cells lines. In the cell lines generated by transfecting the three telomerase deficient cell lines with the mTERC plasmid or with the empty vector (see below), the RD50 were: ~11 µM PALA for KO16 BS and KO16 mTERC, ~13 µM PALA for KO11F6 BS and KO11F6 mTERC, ~14 µM PALA for KO9F6 BS and 16 µM PALA for KO9F6 mTERC, respectively.

Selection of PALA resistant cells
A total of 105 cells per 10 cm dish were seeded in selective medium containing 1 µM dipyridamole and 160, 240 or 320 µM PALA, that is between 8 and 32 times the RD50 value. After 3 weeks, either PALA resistant clones were fixed and counted or were isolated. From WT17 wild-type cells, we isolated 9 clones resistant to 160 µM PALA and one resistant to 240 µM PALA; from KO16 cells 5 clones resistant to 160 µM PALA, 4 clones resistant to 240 µM PALA and 4 clones resistant to 320 µM PALA; from KO9F6 cells 6 clones resistant to 160 µM PALA, 2 clones resistant to 240 µM PALA and 2 clones resistant to 320 µM PALA; from KO11F6 cells, 5 clones resistant to 160 µM PALA, 2 clones resistant to 240 µM PALA and 3 clones resistant to 320 µM PALA. To select PALA resistant cells from the telomerase deficient cells transfected either with mTERC or with the empty plasmid (see below), four 10 cm dishes were seeded with 105 cells in the presence of 1 µM dipyridamole and 160 µM PALA; after three weeks, resistant cells were collected as polyclonal populations.

Chromosome preparation and FISH
Chromosome spreads were prepared according to standard procedures. As expected, none of the immortalized cell lines was diploid anymore. PALA resistant clones were propagated in selective medium and chromosome spreads were prepared after 2 or 3 passages. By FISH, we analyzed 9 clones resistant to 160 µM PALA and one resistant to 240 µM PALA from WT17 cells; 4 clones resistant to 160 µM PALA, 3 clones resistant to 240 µM PALA and 3 clones from 320 µM PALA from KO16 cells; 6 clones resistant to 160 µM PALA, 2 clones resistant to 240 µM PALA and 2 clones resistant to 320 µM PALA from KO9F6 cells; 5 clones resistant to 160 µM PALA, 2 clones resistant to 240 µM PALA and 3 clones resistant to 320 µM PALA from KO11F6 cells. For FISH analysis, slides were pretreated with RNase A (100 µg/ml) for 1 h at 37°C and then with pepsin (1 mg/ml, pH2) for 10 min at 37°C. As probe for slides hybridization, a PAC (P1-derived artificial chromosomes) containing the mouse CAD gene was used (pCAD). For each slide, 200 ng of probe was competed with a 200-fold excess of sonicated mouse DNA. Hybridization was performed over night at 37°C in 50% formamide and washes were performed at 42°C in 50% formamide in 2x SSC. Chromosomes were counterstained with 4'-6-diamidino-2-phenylindole (DAPI, 200 µg/ml). Metaphases were observed using an optical microscope Olympus IX71 equipped with an 100x objective. Images were taken with a digital camera Cool SNAPES (Photometrics) using the MetaMorph software.

Southern blotting analysis
A total of 10 µg of DNA were digested with 50 U of EcoRI for 18 h and separated onto a 0.7% agarose gel. The gel was blotted onto a nylon membrane (Amersham) and the DNA was hybridized with a genomic fragment of 3 kb generated by PCR amplification from the pCAD PAC using primers specific for the CAD gene (fwd 5'-TCATTAGCTGTGGGTTCTGGT-3' and rev 5'-TGGAGCTCCTGCGCTGACGTT-3'). As control for DNA loading, membranes were hybridized with a 618 kb anonymous fragment derived from the mouse chromosome 15; the fragment was generated by PCR with the primers: fwd 5'-CAGCTAGATTACAGCCCCTC-3' and rev 5'-ACAACTCCTTCTCTCCTGTGGAC-3'. Hybridization signals were detected and revealed using a Phosphor-Imager apparatus (Applied Biosystem), and quantified using the Image-Quant software.

Plasmids and transfection
mTERC plasmid was derived from the pBlueScript KS+ plasmid (pBS) and contains the mTERC gene under its promoter together with the gene for puromycin resistance. In parallel, we used the empty vector (pBS-PURO) containing only the gene for puromycin resistance. Before transfection, the two plasmids were linearized with ScaI.

For transfection, 2.8 x 105 cells were seeded in a 6 cm dish. Transfection was performed using FuGene6 (Roche) reagent, according to standard procedures, with 8.4 µg of ScaI linearized plasmids. Transfected cells were selected with 8 µg/ml of puromycin.

RT–PCR
RNA was extracted using Nucleo Spin RNA II kit (Macherey-Nagel), according to the instruction of the supplier. A total of 1 µg of RNA was reverse-transcribed with random primers in the presence and in the absence of reverse transcriptase (Promega); the cDNA was amplified by PCR using primers specific for mouse mTERC (fwd 5'-TCATTAGCTGTGGGTTCTGGT-3' and rev 5'-TGGAGCTCCTGCGCTGACGTT-3'). As internal control, the cDNAs were amplified with primers for mouse G3PDH.

Telomerase assay
Telomerase assay was performed using the TRAPeze kit (Chemicon) with 1 µg of protein extract according to the instruction of the supplier. A total of 1 µg of protein extract was used for each assay. For the KO16 cell line, the extended product was extracted with phenol–chloroform–isoamyl alcohol and precipitated before PCR amplification.

Terminal restriction fragment (TRF) length analysis
In transfected cells, TRFs were analyzed after 9–11 PDs since transfection. Single cell suspensions were mixed with an equal volume of 2% InCert Agarose (Cambrex) to make plugs containing 5 x 105 cells each. Plugs were digested with 50 U each of AfaI and HinfI for 18 h at 37°C. Digested DNA was separated on 1% Ultrapure agarose (Life Technologies) gel in 0.5x TBE maintained at 14°C, using CHEF-DR® II Pulsed Field Electrophoresis System (BIORAD). Separation was performed for 16 h at 6 V/cm at a pulse time of 1–6 s. The gel was blotted onto a nylon membrane (Amersham) and the DNA was hybridized with a 32P-labeled probe for the telomeric TTAGGG repeats. The probe was a mixture of synthetic (TTAGGG)n fragments ranging in size from 1 to 20 kb, prepared as described in (19).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The CAD gene is not amplified in PALA resistant clones from mTERC–/– cells
In rodent cells, the main mechanism for PALA resistance is the amplification of the CAD gene (33,34). Since in primary MEFs gene amplification is undetectable (2,3), we analyzed PALA resistance in telomerase deficient cell lines obtained from embryonic fibroblasts immortalized through the 3T3 protocol (30,31). In particular, we studied one immortalized embryonic fibroblast cell line derived from wild-type mice, one cell line derived from G1 mTERC–/– mice (KO16), and two cell lines derived from G6 mTERC–/– mice (KO9F6 and KO11F6).

Initially, we determined the frequency of clones resistant to 160 µM PALA in each cell line and we found that it was higher in mutant cells compared to wild-type cells (0.3 x 10–4 in wild-type cells versus 1.9 x 10–4 in KO16, 6.9 x 10–4 in KO9F6 and 3.1 x 10–4 in KO9F11). Subsequently, we selected clones resistant to different PALA concentrations (160, 240 and 320 µM PALA) and, by fluorescent in situ hybridization on metaphase spreads, we analyzed the organization of the CAD gene in 10 clones of each cell line (see Materials and methods).

In 9 out of 10 clones isolated from wild-type cells, multiple hybridization signals were detected, indicating that the CAD gene was amplified. In 8 clones, amplification was intrachromosomal (Figure 1A), while in one clone the additional copies of the CAD gene were located on double minutes (Figure 1B). In the majority of the clones, the intrachromosomal amplified regions were organized in ladder-like structures, in which the additional copies of the CAD genes were separated by blocks of heterochromatin (Figure 1A). These chromosomes can originate through BFB cycles with the break preferentially occurring within the heterochromatic region. We then analyzed 30 PALA resistant clones isolated from the telomerase deficient cell lines, 10 from each line and, surprisingly, in none of them we observed extra copies of the CAD gene. On all the metaphases, a hybridization signal attributable to a single copy CAD gene was located on the pericentromeric region of the long arm of chromosome 5 (Figure 1C–E), where the gene is localized.


Figure 1
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  		Fig. 1 FISH analysis of CAD on metaphases prepared from PALA resistant clones. For each partial metaphase, DAPI staining is also shown (lower image). (A) Example of intrachromosomal amplification in a clone from wild-type cells, multiple hybridization signals (red dots) are visible on a chromosome; (B) example of extrachromosomal amplification on double minutes in one wild-type clone; (C–E), hybridization signals due to a single copy CAD in clones isolated from mTERC–/– KO16, KO9F6 and KO11F6 cells, respectively; (F and G) partial metaphases with intrachromosomal CAD amplification in PALA resistant KO16 (F) and KO11F6 (G) cells transfected with the mTERC gene.

 
To check whether PALA resistant clones isolated from telomerase deficient cells carried extra copies of the CAD gene on episomal structures too small to be cytologically detectable, we analyzed the CAD gene copy number in DNA samples prepared from PALA resistant clones by Southern blotting. Blots were hybridized with a probe for the CAD gene (Figure 2A) and with a probe for a random sequence located on chromosome 15 to normalize for DNA loading (Figure 2B). From the analysis of the hybridization patterns shown in Figure 2, we found that in the four PALA resistant clones isolated from wild-type cells, the average number of copy of CAD was from 3 to 4.7 times greater than in parental cells, confirming the presence of additional copies of the CAD gene. In contrast, in none of the PALA resistant clones derived from telomerase deficient cells that we analyzed (six from KO16 and KO9F6 cells and five from KO11F6), Southern hybridization revealed the presence of additional copies of the CAD gene.


Figure 2
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  		Fig. 2 Southern blot hybridization with a probe for the CAD gene (A) or with a random probe for mouse chromosome 15 (B) on DNA samples prepared from PALA resistant clones isolated from WT17, KO16, KO9F6 and KO11F6 cells and from the parental cell lines. The name of each parental cell line is in bold characters; the number in the clone names corresponds to the PALA dose used for selection. The intensities of the hybridization bands obtained with the random probe in each parental cell line and in the corresponding PALA resistant clones have been used to normalize for DNA loading in the different samples and to determine the ratio between the intensity of the CAD hybridization band in each PALA resistant clones and in the corresponding parental cells; this value gives a measure of the relative number of CAD gene copies in the PALA resistant clones.

 
Taken together, these results strongly indicate that, in telomerase knockout cells, CAD gene amplification is inhibited.

CAD gene amplification ability is restored in cells transfected with the mTERC gene
To confirm that the inhibition of CAD gene amplification in telomerase deficient cells was causally related to their genetic defect, we analyzed gene amplification in the same cell lines after complementation with the mTERC gene (28). To this purpose, we transfected the three telomerase deficient cell lines KO16, KO9F6 and KO11F6 with a plasmid containing the mTERC gene and the gene coding for puromycin resistance, or with a control empty vector. Then we selected transfected cells with puromycin and we isolated polyclonal resistant populations. We did not select single clones, because we reasoned that cell lines of polyclonal derivation could better represent the original parental cell lines. In the six cell lines we analyzed mTERC gene expression by RT–PCR and telomerase activity by TRAP assay. As shown in Figure 3A, the expected PCR product corresponding to the expression of the mTERC gene was detected in the mTERC transfected cells but not in the corresponding cells transfected with the empty vector (Figure 3A lanes 5, 9, 13 versus 3, 7, 11). In addition, mTERC expression was associated with the re-establishment of telomerase activity in KO16 mTERC, KO9F6 mTERC and KO11F6 mTERC cells (Figure 3B).


Figure 3
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  		Fig. 3 Analysis of mTERC RNA expression by RT–PCR (A) and of telomerase activity by TRAP assay (B) in mTERC–/– KO16, KO9F6 and KO11F6 cell lines transfected either with the empty plasmid (suffix BS) or with the plasmid containing the mTERC gene (suffix mTERC). In (A) each RNA sample was incubated with reverse transcriptase (+) or, as negative control, in the absence of the enzyme (–). G3PDH was used as internal control. In (B) lane 1: protein extract from a telomerase positive control cell line; {Delta}H: heat inactivated protein extracts; IC: internal control.

 
The telomerase deficient cell lines transfected either with the mTERC plasmid or with the empty vector showed similar PALA sensitivity (see Materials and methods). We isolated polyclonal populations resistant to 160 µM PALA from each transfected cell line and we analyzed CAD amplification by FISH on mitotic chromosomes. As shown in Table I, in KO16 and KO11F6 cells transfected with mTERC 28.2 and 37.6% of cells, respectively, carried amplified CAD genes, while in the corresponding lines transfected with the empty vector, amplification was detected only in 0.3% of the metaphases. In contrast, the frequency of PALA resistant clones was similar in KO16 and KO11F6 cells either transfect with the mTERC plasmid or with the empty vector (2.8 x 10–4 in KO16 BS and 3.9 x 10–4 in KO16 mTERC; 3.1 x 10–4 in KO9F6 BS and 3.1 x 10–4 in KO11F6 mTERC). In most metaphases, the amplified regions were organized in ladder-like structures similar to those observed in wild-type PALA resistant cells (Figure 1F and G).

Thus, restoration of mTERC expression in KO16 and KO11F6 cells is associated with an increased frequency of gene amplification and, in complemented cells, gene amplification occurs through the same mechanisms operating in wild-type cells.

In KO9F6 cells transfected with the mTERC gene, we did not recover PALA resistant cells with CAD gene amplification (Table I).


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  		Table I CAD gene amplification in PALA resistant polyclonal populations isolated from the telomerase deficient cells transfected with the mTERC gene (suffix mTERC) or with the empty vector (suffix BS)

 
Telomere length in telomerase deficient cells and in cells transfected with the mTERC gene
We analyzed the length of the TRFs by Southern blotting in wild-type, G1 and G6 mTERC–/– parental cell lines, as well as in the cell lines obtained by transfection with the mTERC gene or with the empty vector. DNA samples were digested with AfaI and HinfI, separated on an agarose gel by pulse field electrophoresis, transferred onto a nylon membrane, and hybridized with a telomeric probe. As expected, different TRF distributions were observed among the four parental cell lines (Figure 4A). Telomeres were shorter in G1 mTERC–/– cells compared to wild-type cells (Figure 4A, lane 2 versus lane 1), and in G6 mTERC–/– cells compared to G1 cells (Figure 4A, lanes 3 and 4 versus lane 2); in particular, the G6 KO9F6 cell line (Figure 4A, lane 3) showed a narrower distribution of telomere length, with a decrease in the hybridization signal both a the higher and at the lower molecular weights. Since we did not detect gene amplification in any of the telomerase deficient cell lines, the different telomere lengths do not seem to play a role in gene amplification. In addition, we did not observe significant differences in the TRF distribution between each cell line and its counterpart transfected with the mTERC gene or with the empty vector (Figure 4B, lanes 1, 3, 5, 7 versus lanes 2, 4, 6, 8), confirming that telomere length per se does not affect the amplification ability of the telomerase deficient cell lines.


Figure 4
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  		Fig. 4 TRF distribution in DNA samples prepared (A) from parental WT17, KO16, KO9F6 and KO11F6 cell lines and (B) from the same cell lines transfected with the empty plasmid (suffix BS) or with the mTERC plasmid (suffix mTERC).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this paper, we have shown that CAD gene amplification is drastically reduced in mTERC–/– immortalized MEFs. This result was unexpected since mTERC–/– embryonic fibroblasts, either primary or immortalized, are characterized by a high degree of chromosomal instability (28,30). In particular, in telomerase deficient cells, chromatids with critically short telomeres tend to form end-to-end fusions, giving rise to dicentric chromosomes (30). Results obtained in different systems showed that dicentrics can resolve in great variety of chromosomal aberrations (35), among which gene amplification (36). Commitment into BFB cycles of dicentric chromosomes, either derived from the fusion of sister chromatids with non-functional telomeres, or from the fusion of broken sister chromatids, is a frequent mechanism for the genesis of intrachromosomal amplified DNA (14,22,37).

Resistance to PALA is one of the main methods exploited for the analysis of gene amplification, since it is mostly mediated by amplification of CAD, the drug target gene (33). In agreement with this expectation, in 90% of the PALA resistant clones obtained from wild-type immortalized MEFs, we found that PALA resistance was due to amplification of the CAD gene. In all clones but one, amplification was intrachromosomal and likely due to BFB cycles. In most amplified chromosomes, the extra copies of the CAD gene were separated by heterochromatic blocks (Figure 1), suggesting that the pericentromeric heterochromatic region of chromosome 5 is a preferential break site during BFB cycles. In contrast to what was observed in wild-type cells, in none of the PALA resistant clones isolated from the three telomerase deficient cell lines, both of the first and of the sixth generation, we found amplified copies of the CAD gene. This observation indicates that, in mTERC–/– cells, PALA resistance is achieved through mechanisms other than gene amplification. We can exclude that PALA resistance is due to a reduced uptake of the drug, since all the three telomerase deficient cell lines are more sensitive to PALA than wild-type cells; in fact, the dose of PALA reducing growth to 50% is around 20 µM PALA in wild-type cells and 10 µM PALA in telomerase deficient cells (data not shown). PALA resistance in telomerase deficient cells could be due to mutations in the CAD gene leading to a more active enzyme, to variations in CAD level of expression, or to mutations in other genes affecting the intracellular nucleotide pool. Lack of CAD amplification was described in a few PALA resistant clones isolated from Chinese hamster cells (38), and more frequently in resistant cells from human cell lines (39).

In the telomerase deficient cell lines, the frequency of PALA resistant clones was higher than in wild-type cells, suggesting that, besides the decrease in the frequency of gene amplification, proneness towards other kinds of mutations characterizes mTERC–/– cells. We are not aware of data concerning mutation frequencies in telomerase deficient cells in higher eukaryotes. An increased frequency of mutations in the CAN1 gene was reported in Saccharomyces cerevisiae cells characterized by telomere instability because of the deletion of the est1 gene (40); in these cells, the higher frequency of mutations was due to chromosomal rearrangements driven by telomere instability. We cannot exclude that chromosomal rearrangements can play a role in the induction of mutations at loci involved in PALA resistance; however, it is difficult to hypothesize that they are involved in the genesis of mutations of the CAD gene, since the CAD bearing chromosome does not appear rearranged in PALA resistant cells. Although, our work does not allow us to draw definite conclusions about mutation frequency in telomerase deficient cells, this potentially interesting phenomenon will warrant further investigation.

The causal relationship between mTERC deficiency and absence of gene amplification was demonstrated by the analysis of deficient cells in which mTERC expression had been restored by transfection with the mTERC gene. In two out of the three transfected lines, we could find a significant fraction of PALA resistant cells bearing CAD gene amplification (~30%), while in mock transfected cells amplification was present only in 0.3% of the cells. It is unlikely that the increased appearence of metaphases with CAD amplification in cell with mTERC restored was due to decreased frequency of PALA resistant cells without CAD amplification, since in the the mock transfected populations, the frequency of PALA resistant clones was similar to that observed in the cell lines in which the genetic defect had been complemented. This observation makes improbable that CAD amplified cells were not detected in the telomerase deficient cell lines because overwhelmed by resistant cells without amplification. The striking different behavior between the telomerase deficient cells transfected with mTERC and those mock transfected, clearly indicates that restoration of mTERC expression is associated with the acquisition of gene amplification ability. The observation of a similar organization of the amplified chromosomes in wild-type cells and in mTERC complemented telomerase deficient cells suggests that in both cases amplification occurs through the same mechanisms.

O'Hagan et al. (41) have recently shown that, compared to tumors found in mice with intact telomeres, tumors arising in late generation mTERC–/– mice have a higher level of genome instability, with amplifications and deletions, which could be induced by telomere dysfunction and associated BFB cycles. In this system, the observation of amplified DNA can be the result of positive selection of the cells at the initial phases of gene amplification. In fact, most of the chromosome regions with abnormal dosage contain cancer-relevant genes, whose increased expression confers proliferative advantage and could drive tumor development. In contrast, in our system, as discussed in the paragraph below, telomere length does not seem to play a role in the initiation of gene amplification and the rare cells that begin CAD amplification do not have any advantage compared to those that acquire resistance to PALA through other mechanisms. Thus, our system could allow us to highlight the low intrinsic amplification propensity of telomerase deficient cells.

As expected, the three telomerase deficient cell lines we studied had shorter telomeres than wild-type cells, and showed different patterns of telomere length distributions. Despite the differences in telomere length, we did not recover amplified cells from any of the cell lines, suggesting that telomere length per se is not relevant for gene amplification in this cellular system.

How can mTERC influence gene amplification? We can propose different answers to this question. First of all, telomerase RNA is part of the telomerase enzyme and is essential for telomerase activity; telomerase activity could play a direct role in gene amplification by stabilizing the amplified chromosomes. According to the BFB cycle mechanism, which is the primary mechanism used by the immortalized wild-type MEFs, amplified chromosomes become stable when a telomere is added to the broken ends (19). In the absence of telomerase activity stabilization of broken ends is impaired, thus the probability to recover amplified chromosomes can also drop down. Alternatively, and or in addition, telomerase could play an indirect role in gene amplification. It has been shown that telomerase can promote tumorigenesis and can modulate the expression of growth-controlling genes in a way independent of its function in telomere maintenance (42–44); analogously, telomerase could intervene in gene amplification by modifying the expression of genes taking part to the process. Finally, it cannot be excluded that telomerase RNA per se is relevant for gene amplification through interactions with other molecules. In humans, for example, hTERC can bind to dyskerin and in the absence of this binding, because of mutations in dyskerin or in hTERC, the rare disease dyskeratosis congenita is developed (45).

In conclusion, our work indicates a role for telomerase in gene amplification. In this regard, we would like to remind that cells bearing amplified genes have never been selected from primary cells; considering that telomerase is virtually absent in these cells, our results suggest another feature of normal cells that can contribute to the inhibition of gene amplification.


   Acknowledgments
 
Work was supported by EU grant FIGHT-CT 2002-217 and by the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (FIRB RBNE01RNN7 and RBAU01ZB78). P. R. was a recipient of a fellowship of the ‘Adriano Buzzati Traverso’ Foundation.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 3, 2006; revised August 18, 2006; accepted August 21, 2006.


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