Telomere dysfunction and telomerase reactivation in human ... - Nature

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Oncogene (2002) 21, 8262 – 8271 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc

Telomere dysfunction and telomerase reactivation in human leukemia cell lines after telomerase inhibition by the expression of a dominant-negative hTERT mutant François Delhommeau1, Antoine Thierry1, Danie`le Feneux2, Evelyne Lauret1, Edwige Leclercq3, Marie He´le`ne Courtier3, Françoise Sainteny1, William Vainchenker1 and Annelise Bennaceur-Griscelli*,1,3 1

INSERM U362, PR-1, Institut Gustave Roussy, 39/53 rue Camille Desmoulins, 94805 Villejuif, France; 2Service d’He´matologie et de Cytogeńe´tique, Hoˆpital de Biceˆtre, 78 rue du Geńe´ral Leclerc, 94270 Le Kremblin Biceˆtre, France; 3Department of Clinical Biology, Service d’He´matologie Biologique, Institut Gustave Roussy, 39/53 rue Camille Desmoulins, 94805 Villejuif, France

As activation of telomerase represents a key step in the malignant transformation process, experimental models to develop anti-telomerase drugs provide a rational basis for anticancer strategies. We analysed the short and longterm efficacy of a stably expressed dominant-negative mutant (DN) of the telomerase catalytic unit (hTERT) in UT-7 and U937 human leukemia cell lines by using an IRES-e-GFP retrovirus. As expected, telomerase inactivation resulted in drastic telomere shortening, cytogenetic instability and cell growth inhibition in all e-GFP positive DN clones after 15 – 35 days of culture. However, despite this initial response, 50% of e-GFP positive DN clones with short telomeres escaped from crisis after 35 days of culture and recovered a proliferation rate similar to the control cells. This rescue was associated with a telomerase reactivation inducing telomere lengthening. We identified two pathways, one involving the loss of the DN transgene expression and the other the transcriptional up-regulation of endogenous hTERT with persistence of the DN transgene expression. Although this second mechanism appears to be a very rare event (one clone), these findings suggest that genomic instability induced by short telomeres after telomerase inhibition might enhance the probability of activation or selection of telomere maintenance mechanisms dependent on hTERT transcription. Oncogene (2002) 21, 8262 – 8271. doi:10.1038/sj.onc. 1206054 Keywords: telomere; telomerase; leukemia; retrovirus; cell death; therapy

Introduction Telomeres are dynamic DNA-protein complexes (Blackburn, 2001) that cap the ends of linear

*Correspondence: A Bennaceur-Griscelli; E-mail: [email protected] Received 3 July 2002; revised 13 September 2002; accepted 17 September 2002

chromosomes consisting of TTAGGG repeats (de Lange et al., 1990) and telomere-binding proteins (Broccoli et al., 1997; Chong et al., 1995). Telomeres protect the chromosome ends from degradation, recombination and DNA repair activities (Bailey et al., 1999; d’Adda di Fagagna et al., 1999; Samper et al., 2000). In addition, telomere length is progressively reduced with cell divisions (Blasco et al., 1997; Harley et al., 1990), due to the ‘end-replication problem’ (Hastie et al., 1990) and the putative exonuclease activity in the CA-rich strand (Makarov et al., 1997). Telomere shortening in aging cells induces replicative senescence (Morin, 1997). When telomeres reach a critical size, chromosomes become unstable and undergo end-to-end fusions, DNA fragmentation, and mutations (Blasco et al., 1997; Gisselsson et al., 2001). Conversely, telomere length is stable in cells with long-lived replicative life spans, such as germ line and embryonic cells, and in immortalized and tumor cells (Wright et al., 1996). Telomerase activation is the major mechanism that maintains telomere integrity (Morin, 1989). The human telomerase reverse transcriptase (hTERT) is an RNA-dependent DNA polymerase which synthesizes telomeric DNA using the human telomerase RNA (hTR) as a template (Colgin and Reddel, 1999; Feng et al., 1995; Morin, 1989; Nugent and Lundblad, 1998). Unlike germline and embryonic cells, somatic cells do not have telomerase activity, with the exception of regenerative tissues such as hematopoietic stem cells (Chiu et al., 1996; Morrison et al., 1996) or lymphocytes (Hiyama et al., 1995). In contrast, most malignant cells are characterized by an increased telomerase activity (Meyerson et al., 1997; Raymond et al., 1996). Enhanced telomerase activity represents an important step in the transformation process of human cells, as the combined expression of hTERT, SV40 large T antigen and H-Ras results in direct conversion from normal to tumor cells (Hahn et al., 1999a). In addition, acquisition of telomerase activity may be associated with escape from senescence (Bodnar et al., 1998; Counter et al., 1998a; Pendino et al., 2001). These findings validate

Telomerase inhibition in human leukemia cell lines F Delhommeau et al

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telomerase as an important drug target for cancer therapy. Telomerase inhibitors are considered as promising agents for a wide variety of human malignancies. Impairing telomerase activity in human solid tumors by peptide nucleic acid or 2’-O-MeRNA (Herbert et al., 1999), chemical compounds (Damm et al., 2001), anti-sense oligonucleotides (Kondo et al., 1998), mutant hTR (Kim et al., 2001), hTERT dominantnegative (DN) mutants (Hahn et al., 1999b; Zhang et al., 1999), or by G-quadruplex DNA ligands (Riou et al., 2002) results in cell growth inhibition and loss of tumorigenicity. Surprisingly, ‘revertant’ clones were selected in one murine tumor cell line after telomerase inhibition by a DN mutant, suggesting differences in telomerase regulation between human and murine cells (Sachsinger et al., 2001). Moreover, lessons learned from knock-out mice have to be considered since carcinogenesis is accelerated when both murine telomerase RNA and p53 genes are inactivated (Chin et al., 1999). In addition, the possibility of resistance emerging from indirect telomerase-based therapies has been recently observed in acute promyelocytic leukemia cells treated by retinoic acid (Pendino et al., 2002). Thus it is possible that genetic instability induced by short telomeres might promote the selection of resistant sub-clones in human tumor cells subjected to a long term anti-telomerase strategy. As the possibility that tumor cells might overcome telomerase inhibition has not been carefully evaluated in human tumors, we investigated this hypothesis in this work by studying the susceptibility of telomerase positive human leukemia cell lines to growth and death after short and longterm telomerase inactivation by a DN-hTERT mutant (Hahn et al., 1999b). We studied the effects of telomerase inactivation in the myelomonocytic U937 cell line (Harris and Ralph, 1985) and in the UT-7 megakaryocytic cell line (Komatsu et al., 1991). We show that after an initial response defined by a critical telomere shortening, growth arrest and massive cell death, long-term culture results in the emergence of surviving clones, which escape from crisis by telomerase reactivation. Two mechanisms are responsible for this reactivation: the first one, occurring in both cell lines, involves the loss of DN transgene expression following telomere dysfunction and the second one, exclusively observed in one U937 DN clone, involves the increase in endogenous hTERT transcription with the persistence of the DN transgene expression. Results DN inhibits telomerase activity and induces telomere shortening in leukemia cell lines U937 and UT-7 cells were transduced by the control vector Mig-R and Mig-R DN-hTERT retroviruses (Figure 1). After three days of culture, e-GFP positive cells were sorted by flow cytometry and seeded at one cell per well. Telomerase activity was measured in proliferating clones when they reached 1006103 cells

Figure 1 Schematic representation of the Mig-R and Mig-R-DN constructs

per well. A dramatic decrease in telomerase activity was observed in 77% of U937 DN clones (31 out of 40) and 63% of UT-7 DN clones (47 out of 75), compared to control vector clones (Figure 2a). The incidence of telomerase negative clones in DN clones was not different between clones with low and high expression of e-GFP (data not shown). Consequences of telomerase inhibition on telomere length were evaluated by Southern blot and Q-FISH in transduced U937 and UT-7 clones after 25 – 35 and 20 – 25 PDs respectively (Figure 2b). Telomere length of the clones transduced by the empty vector was not significantly different from uninfected parental cell lines (Figure 2b, lanes 1, 2, 3). During expansion, all DN U937 and UT7 clones exhibiting initially a reduced telomerase activity remained e-GFP positive and TRAP negative (data not shown) indicating the stability of the transgene expression. Progressive telomere shortening was observed in all these clones, reaching values around 2 kb (Figure 2b, lanes 4, 5, 6). Q-FISH analyses confirmed these data as a net decrease in telomere fluorescence intensity was observed in DN clones when compared to control clones (Figure 2c). DN affects cell growth and triggers cytogenetic instability and cell death Clonogenicity (i.e. frequency of proliferating clones from initial seeded cells) of DN cells was similar to control cells in U937 and UT-7 cell lines (25 out of 60 control vector clones versus 51 out of 120 DN clones and 102 out of 180 control vector clones versus 154 out of 240 DN clones respectively), indicating the absence of cytotoxicity of DN in first mitoses. After 2 weeks of culture, 82% of DN UT-7 clones had stopped growing compared to 55% of control UT-7 clones. In U937 clones, a delayed effect relative to initial longer telomeres in this cell line (5.5 kb compared to 3.5 kb in UT-7 cell line) was observed. The loss of viability induced by telomere shortening was confirmed by an increased rate of dead cells when analysing propidium iodide incorporation in both U937 and UT-7 DN clones (Figure 3a). These clones displayed senescent features such as enlarged cells and granular morphology (data not shown). Since genetic instability is a direct consequence of shortened telomeres’ dysfunction, we performed cytogenetic analyses. Additional end-toOncogene


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Figure 2 Telomerase inhibition and telomere shortening in DN clones. (a) Inhibition of telomerase activity by DN in U937 and UT-7 clones. TRAP assays were carried out in HEK 293 as positive control (lanes 1, 2), in representative U937 control vector clone (lanes 3, 4), U937 DN clone (lanes 5, 6), UT-7 control vector clone (lanes 7, 8) and UT-7 DN clone (lanes 9, 10). T: TRAP products; IC: Internal control PCR products; H+ +: heat treatment of samples before TRAP assay; H7 7: no heat treatment. (b) TRF analysis in U937 and UT-7 clones respectively 25 – 35 and 20 – 25 PDs after transduction with control and DN vectors. Lane 1: uninfected cells; Lanes 2 – 3: control vector clones; Lanes 4 – 6: representative DN clones. Left margin: molecular size (kb). (c) Q-FISH analysis of telomere fluorescence in interphase cells from U937 and UT-7 control and DN clones. DAPI-stained nuclei and Cy-3-labeled telomeres appear respectively in blue and red

end fusions with dicentric, multicentric and ring chromosomes were observed in both U937 and UT-7 DN clones with shortened telomeres (Figure 3b) when Oncogene

compared to control vector clones. In the U937 cell line, no sign of cytogenetic instability was found in control vector clones (n=29 metaphases) whereas 36%


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stable telomerase activity measured by semi-quantitative TRAP assay was always maintained in e-GFP positive control clones (Figure 4b). Analysis of telomerase activity and telomere length in surviving clones with increasing PDs demonstrated the reversibility of telomerase inhibition followed by telomere lengthening in UT-7 and U937 DN clones (Figure 4c). However, among these surviving clones, a sole U937 clone (DN95) exhibited a drastic increase in telomerase activity, up to 400% of the control vector clones’ values after 80 PDs, leading to a dramatic elongation of telomeres (Figure 4c). In order to accurately quantify this apparent increase in telomerase activity, we performed a real-time quantitative TRAP modified protocol that confirmed a 2.8+0.3-fold increase in telomerase activity in the U937 DN95 clone in comparison to three control vector clones (Figure 4d). Molecular mechanisms involved in telomerase reactivation

Figure 3 Cellular consequences of telomere shortening in U937 and UT-7 clones at 25 – 35 and 20 – 25 PDs respectively after transduction with DN or the control vector. (a) PI incorporation histograms: the percentage indicates the cell fraction with sub-G1 DNA levels. (b) Representative metaphases of DN U937 (standard coloration) and DN UT-7 (Banding G) clones harboring dicentric or multicentric chromosomes. Arrows indicate the centromeres of dicentric and multicentric chromosomes

of DN clones’ metaphases (n=30) harbored dicentric, multicentric or ring chromosomes. Although uninfected UT-7 cell line and UT-7 control vector clones were found to exhibit a basal cytogenetic instability, we observed a greater number of metaphases with dicentric, multicentric or ring chromosomes in DN clones compared to control vector clones (70% versus 20%, respectively) demonstrating an increase in genetic instability. Reactivation of telomerase activity in DN clones at late passage leads to rescue from crisis In order to study the long-term efficacy of telomerase inactivation in transduced DN e-GFP positive clones, we maintained slowly growing DN clones in culture and monitored their outcome. Among these clones, 5 out of 11 U937 and 10 out of 18 UT-7 clones underwent massive cell death leading to complete extinction. Interestingly, some DN clones continued to survive despite an initially abolished telomerase activity, very short telomeres and cytogenetic instability. Indeed, 55% (6 out of 11 clones) of U937 and 44% (8 out of 18 clones) of UT-7 DN clones recovered a normal cell growth similar to control clones after 35 and 55 days of culture, respectively (Figure 4a). Escape from crisis was attributed to telomerase reactivation in all residual survival DN e-GFP positive clones after the period of growth inhibition and cytogenetic instability induced by critical telomere shortening. In contrast, a

Except the UT-7 DN14 and U937 DN95 clones, all other surviving DN clones became e-GFP negative, suggesting the loss of transgene expression (Figure 5a). The DN transgene loss was confirmed by PCR in the eGFP negative clones (data not shown). The transgene/ IRES junction was analysed by RT – PCR in the clones remaining e-GFP positive. In contrast to UT-7 DN14 clone which had lost transgene expression despite the persistence of the e-GFP expression, DN mRNA was still present in U937 DN95 clone (Figure 5b). Genomic PCR demonstrated the deletion of the DN sequence in UT-7 DN14 clone without alteration of the IRES/GFP sequence (data not shown), which may explain the persistence of the GFP expression. In order to understand the increase in telomerase activity in U937 DN95 clone, we performed a semi-quantitative RT – PCR to calculate the relative quantity of endogenous hTERT and DN mRNAs. The presence of the NruI site in the mutant cDNA discriminates the endogenous hTERT from the mutant DN (Figure 6a). The relative percentages of endogenous and DN mRNAs were estimated and their respective quantities were calculated after quantification of total endogenous and DN-hTERT transcripts using real-time quantitative RT – PCR (Figure 6b). A significant threefold increase in hTERT transcription level (P=0.03, n=3) was observed in the U937 DN95 clone when compared to three control vector clones. As amplification of hTERT gene has recently been described in human tumors (Zhang et al., 2000) we analysed if an increase in hTERT gene copy number had occurred in the U937 DN95 clone secondary to chromosomal instability. No hTERT amplification was observed as illustrated by FISH analysis (Figure 6c). Discussion Several strategies to inhibit telomerase have been reported in various tumor types but not in leukemias with successful in vitro and in vivo responses (Damm et Oncogene


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Figure 4 Cell growth and reactivation of telomerase activity. (a) Cell growth curves of U937 and UT-7 clones: control vector clones appear as gray lines and DN clones as black lines. {Clone extinction after massive cell death. Note the recovery of normal cell growth after transient growth inhibition in U937 DN95 (DN95?) and UT-7 DN14 (DN14?) clones. (b) Telomerase reactivation in DN clones: telomerase activity was measured in U937 and UT-7 clones 30 days (black histograms) and 55 days (gray histograms) after transduction with control vector (control) or DN-hTERT (DN). *Clone extinction before 55 days. TRAP results are expressed as percentages of average control vector clones’ activity. (c) Semi-quantitative measurement of telomerase activity (histograms) and telomere length analysis by TRF or Q-FISH (curves) of UT-7 DN14 and U937 DN95 clones were performed with increasing PDs. TRAP results are expressed as percentages of average control vector clones’ activity. Day 0 measurements of telomere length and telomerase activity were performed using uninfected UT-7 and U937 cell lines. (d) Quantification of telomerase activity in U937 control and DN95 clones performed by real time modified TRAP assay as described in methods. One arbitrary unit (a.u) is defined as the telomerase activity of 1 ng of HL60 protein extract

al., 2001; Hahn et al., 1999b; Herbert et al., 1999; Kim et al., 2001; Kondo et al., 1998; Riou et al., 2002; Zhang et al., 1999). In this study, we analysed the short and long term consequences of an anti-telomerase strategy targeting hTERT in UT-7 and U937 human leukemia cell lines. We show that the expression of the DN-hTERT mutant was efficient with expected effects such as telomere shortening inducing cell growth inhibition, cell death, senescence and chromosomal instability. However, this initial response was followed by the emergence of some clones, which escaped from crisis in long-term cultures. The reversion of telomerase inhibition was due to the reactivation of telomerase by the loss of the transgene or by the up-regulation of endogenous hTERT mRNA transcription with continued expression of the transgene. Growth arrest and cell death, as cellular consequences of telomere shortening achieved by telomerase inhibition, were observed in both UT-7 and U937 leukemia clones, after a delay depending on initial Oncogene

telomere size. This outcome is in agreement with those reported by others in solid tumors (Hahn et al., 1999b; Herbert et al., 1999; Kim et al., 2001; Kondo et al., 1998; Zhang et al., 1999). Growth inhibition is the constant event observed in all studies, independent of the mean of telomerase inhibition and the tumor type. The delayed effect of telomerase inhibition depending on initial telomere length suggests that telomerase inhibition has to be sufficiently long to allow tumor cells to reach critically shortened telomeres and the expected crisis. Moreover, an efficient and permanent telomerase inhibition appears to be critical for the success of this approach regarding the potential risk of additional genetic alterations induced by telomere dysfunction (Gisselsson et al., 2001). The striking observation in our study was the appearance of long-term surviving clones overcoming the telomerase inhibition. In both cell lines, 50% of the clones, lacking initially telomerase activity, regained viability and returned to normal growth. Most of these


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Figure 5 Loss of DN transgene. (a) e-GFP fluorescence intensity in representative U937 and UT-7 uninfected, control and DN clones after 20 PDs (filled histograms) and 45 PDs (open histograms). In contrast to U937 DN12 and UT-7 DN8 clones, which had lost e-GFP expression at PD 45, U937 DN95 and UT-7 DN14 clones remained e-GFP positive, suggesting the persistence of transgene expression. (b) Loss of DN transgene expression in UT-7 DN14 clone but not in U937 DN95 clone: DN-hTERT/IRES junction RT – PCR in control clones (Control), UT-7 DN14 and U937 DN95 clones using Mig-R1-DN plasmid (pDN) as positive control

clones escaped by the loss of DN-hTERT expression. This event clearly occurred simultaneously with the critical phase with very short telomeres and genetic instability. The loss of the transgene was never observed in control clones nor in wild-type hTERT transduced clones (data not shown) during the period of expansion. Thus, this event may be considered as a consequence of the combination of the selection pressure during long-term expansion and the telomere dysfunction induced by the telomere shortening and not of the initial telomere-independent genomic instability, especially in UT-7 cell line. Genomic alterations, such as DNA fragmentations and mutations (Gisselsson et al., 2001), induced at late passage by telomere dysfunction could contribute to alter the transgene

and to abrogate its DN function. It might be possible that these chromosomal instability events depend on the non-functional status of p53 in U937 cell line (Dou et al., 1995). However, this mechanism of rescue remains specifically dependent on the strategy used to inhibit telomerase and clearly limits the efficacy of a gene therapy approach. In addition, this observation is of potential interest as clones that had lost transgene expression mimic an interruption of the telomerase inhibition. A hypothesis which has to be confirmed by in vivo studies arises from these observations: an inadequate arrest of telomerase inhibition in the period of chromosomal instability might select clones with a phenotype distinct from initial tumor cells due to additional genomic disorders. Oncogene


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Figure 6 Overexpression of hTERT in U937 DN95 clone. (a) Semi-quantitative RT – PCR encompassing reverse transcriptase region of hTERT transcripts (7), followed by NruI digestion (+) using b2 microglobulin (b2 M) as a housekeeping gene. Left margin: endogenous hTERT unspliced transcript RT – PCR products (749 bp), a and b spliceoform RT – PCR products (spliced). Right margin: NruI digestion of DN PCR products (749 pb) migrate as 427 and 322 pb bands. Efficiency of NruI digestion was demonstrated using PCR products from Mig-R1 DN plasmid (pDN). (b) Semi-quantitative (left panel) and real-time quantitative RT – PCRs (right panel) or hTERT transcripts in U937 control and DN95 clones. Hatched histogram represents the quantification result of total unspliced transcripts, comprising endogenous hTERT mRNA and DN mRNA. *Calculated endogenous hTERT value using the formula: endogenous hTERT=qH6(dH/dS)/(H/S) where qH is the amount of endogenous hTERT+DN transcripts as determined by real-time quantitative RT – PCR, dH and dS are the respective fluorescence intensities of 749 bp and spliced bands after digestion by NruI, H and S are the respective fluorescence intensities of 749 bp and spliced bands before digestion by NruI. (c) FISH analyses of hTERT gene copy number in U937 clones in representative metaphases (M) and interphases (I) showing four copies of telomerase gene in both conditions. Blue, DNA stained with DAPI. Green, hTERT gene

Interestingly, we isolated one initially responsive clone (U937 DN95) which rescued from crisis by a drastic reactivation of telomerase despite the stable expression of DN transgene. Since the catalytic site of Oncogene

the hTERT mutant was still altered at the DNA level, we can suppose that the DN protein was still functional. In contrast to a recent work in HEK 293 cells (Zhang et al., 1999), we found an increase in


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endogenous hTERT mRNA. A similar mechanism was also recently reported in murine RenCa clones subjected to a mTERT mutant (Sachsinger et al., 2001). Whether this mechanism depends on a TERT component targeting strategy or may occur in alternative modes of telomerase inhibition remains to be explored. Nevertheless, the increase in endogenous hTERT sub-units may have overcome the dominant negative effect in accordance with the putative mechanism of telomerase inactivation by the DNhTERT mutant (Arai et al., 2002). When comparing hTR expression in the U937 DN95 clone and the U937 DN12 clone, which had lost the transgene expression, with the control clones, we did not observe any significant difference (data not shown), which is consistent with the fact that hTERT is the limiting component for telomerase activity (Counter et al., 1998b). It is now well established that c-Myc or Sp1 are involved in the up-regulation of hTERT at the transcription level (DePinho et al., 1991; Kyo et al., 2000). However, RT – PCR and Western blot analyses of c-Myc and Sp1 expression in the U937 DN95 clone revealed no difference with the control vector clones (data not shown). Furthermore, we did not find any hTERT gene amplification as previously described in various tumor cells (Zhang et al., 2000). Nevertheless one can speculate that molecular events leading to endogenous telomerase over-expression have been either induced or selected by telomerase inhibition consequences since such an up-regulation did not occur in control vector clones. This finding suggests that antitelomerase strategy, targeting hTERT, could select rare variant sub-clones enhancing endogenous telomerase to by-pass the crisis. Although this kind of rescue seems to be a rare event, we cannot formally exclude that other clones were able to rapidly evade the inhibitory mechanism, as telomerase inactivation in output eGFP-positive clones was partially observed in 60 – 80% of input clones. In conclusion, we show the potential interest of the anti-telomerase strategy in human leukemia. Moreover, we demonstrate the possible emergence of human leukemia cells overcoming telomerase inhibition by a transcriptional up-regulation of endogenous hTERT. Thus, the appearance of variant clones has to be taken into account for the strategy based on telomerase inhibition. The combination of two telomerase inhibitors targeting distinct elements of telomerase complex or its substrate (Riou et al., 2002) and the association with synergistic therapies such as radiotherapy (Goytisolo et al., 2000; Wong et al., 2000) or chemotherapy (Lee et al., 2001) may contribute to limit the emergence of such resistant sub-clones. Materials and methods Cell lines The U937 myelomonocytic and UT-7 megakaryocyte human leukemia cell lines were cultured in alpha MEM medium supplemented with 10% FCS (Invitrogen, Cergy-Pontoise,

France) and 10 ng/ml GM-CSF (Novartis, Nanterre, France) for the UT-7 cell line. Plasmids The Murine Stem Cell Virus retroviral vector Mig-R1 containing encephalomyocarditis virus (ECMV) internal ribosomal entry sequence (IRES) and green fluorescent protein (e-GFP), was kindly provided by Warren S Pear (University of Pennsylvania, Philadelphia, PA, USA). MigR2 plasmid was constructed by replacing ECMV-IRES by Vascular Endothelial Growth Factor IRES. Mig-R-DN vectors were constructed by subcloning the cDNA sequence of the DN-hTERT mutant from pBABE-puro-DN plasmid provided by Robert A Weinberg (MIT, Cambridge, MA, USA) (Hahn et al., 1999b) into either Mig-R1 or Mig-R2 (Figure 1). Transfections and infections Retroviral supernatants of control and DN vectors were prepared as previously described (Pendino et al., 2001) for further infections of U937 and UT-7 cell lines in the presence of 4 mg/ml of Polybrene (Sigma-Aldrich, Saint Quentin Fallavier, France). E-GFP positive cells were sorted 2 days later by flow cytometry (FACSvantage, Becton Dickinson, Le Pont de Claix, France), according to a low or a high intensity of fluorescence, and seeded at one cell per well in a 96-well plate. The constant expression of e-GFP was controlled twice a week by flow cytometry during all the period of cell expansion. Telomerase activity assays Telomerase activity was measured by a modified TRAP assay, using TRAPeze kit (Intergen, Oxford, UK). Semi quantitative measurement of telomerase activity was performed after polyacrylamide gel electrophoresis using the Storm 840 instrument (Amersham Lifescience, Bondoufle, France). Real-time quantitative TRAP using ABI PRISM 7700 Detection System (Perkin Elmer Applied Biosystems) was performed as described (Hou et al., 2001). The standard curve was established using serial dilutions of HL60 protein extract in CHAPS buffer. One arbitrary unit (a.u) was defined as the telomerase activity of 1 ng of HL60 protein extract. Telomere length analysis Genomic DNA was digested with 10 units each of HinfI and RsaI (Roche, Meylan, France), underwent a 0.7% agarose gel electrophoresis and was transferred onto a nylon membrane for further hybridization to a digoxigenin-labeled probe (TTAGGG)3. Revelation of TRF was performed by chemiluminescence detection. Telomere length was also evaluated by Q-FISH using a Cy3 labeled PNA probe (Dako, Glostrup, Denmark). Cells were hybridized with the probe as previously described (Lansdorp et al., 1996) counterstained with DAPI and mounted. Thirty interphasic nuclei per slide were analysed using a Zeiss Axiophot fluorescence microscope. CCD camera captured nuclei were analysed using Smart Capture Software (Vysis, Voisins le Bretonneux, France) and quantitative analysis of image was performed with Quips IP LAB software. Average results of telomeric fluorescence were expressed as fluorescence intensity arbitrary units. Oncogene


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Cellular assays Cell growth and viability were determined using trypan blue. DNA content was analysed by flow cytometry using 25 mg/ml propidium iodide (PI) (Sigma) in 0.1% NP40 citrate buffer with 0.5 mg/ml of RNAse A (Boehringer). Cytogenetic analysis and in situ hybridization study of hTERT gene copy number Standard cytogenetic techniques were used for chromosome preparations. Metaphases were analysed in giemsa and G or C banding were performed when sufficient material was available. Ten to twenty metaphases per clone were analysed. Cytogenetic instability was evaluated by the numeration of metaphases with clearly distinguishable dicentric, multicentric and ring chromosomes. HTERT gene copy number was studied by FISH. The BAC clone 518C13 was kindly provided by S Burns (Beatson Laboratories, University of Glasgow, Glasgow, UK). Briefly, the probe was prepared by labeling the BAC DNA with digoxigenin using Dig Nick translation Kit (Roche) and hybridization was performed as previously described (Bryce et al., 2000). RT – PCR analyses The expression of endogenous and exogenous hTERT mRNAs was analysed by RT – PCR with primers specific for hTERT (5’-CTGTCGGAAGCAGAGGTCAG-3’ and 5’CTCCATGTCGCCGTAGCACA-3’), hTERT/IRES junction (5’-ATCCTCCTGCTGCAGGCGTA-3’ and 5’-GAGCAA-

TCTCCCCAAGCCGT-3’), and b2-microglobulin (b2-M) housekeeping gene as external standard (5’-TCCTGAAGCTGACAGCATTCG-3’ and 5’-TCCTAGGAGCTACCTGTGGAG-3’). Total RNA was extracted by RNA-B (Q-Biogen, Illkirch, France) and reverse transcribed using MuLV reverse transcriptase (Perkin Elmer Applied Biosystems). To discriminate endogenous hTERT from DN, RT – PCR products were digested by NruI (New England Biolabs, MA, USA). Real-time quantification of total unspliced hTERT transcripts was performed using the LightCycler TeloTAGGG hTERT quantification kit (Roche). Serial dilutions of Mig-R1-hTERT plasmid were used as standards.

Acknowledgments We are grateful to S Stewart and RA Weinberg and to WS Pear for providing the constructs, to S Burns for providing the BAC clone and to F Forestier for the use of the LightCycler. We thank H Raslova and P Foliot for FISH techniques and J Dando for critically reading the manuscript. This work was supported by funds from the Institut National de la Sante´ et de la Recherche Me´ dicale (INSERM), grants from the Association pour la Recherche contre le Cancer (ARC no 5606 and 7364), from Institut Federatif de Recherche of Institut Gustave Roussy (IFR 2000), from the Fondation de France (no 98001281) and from Universite´ Paris-Sud (BQR 2000 no CR112). F Delhommeau was supported by INSERM and A Thierry by the Ligue contre le Cancer.

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