Telomere Dysfunction and Telomerase Reactivation in Human Leukemia Cell Lines After Telomerase Inhibition by the Expression of a Dominant-Negative Htert Mutant

Telomere dysfunction and telomerase reactivation in human leukemia cell lines after telomerase inhibition by the expression of a dominant-negative hTERT mutant

Franc¸ois Delhommeau1, Antoine Thierry1, Danie` le Feneux2, Evelyne Lauret1, Edwige Leclercq3, Marie He´ le` ne Courtier3, Franc¸oise Sainteny1, William Vainchenker1 and Annelise Bennaceur-Griscelli*,1,3

1INSERM U362, PR-1, Institut Gustave Roussy, 39/53 rue Camille Desmoulins, 94805 Villejuif, France; 2Service d’He´matologie et de Cytoge´ne´tique, Hoˆpital de Biceˆtre, 78 rue du Ge´ne´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 chromosomes consisting of TTAGGG repeats (de malignant transformation process, experimental models Lange et al., 1990) and telomere-binding proteins to develop anti-telomerase drugs provide a rational basis (Broccoli et al., 1997; Chong et al., 1995). Telomeres for anticancer strategies. We analysed the short and long- protect the chromosome ends from degradation, term efficacy of a stably expressed dominant-negative recombination and DNA repair activities (Bailey et mutant (DN) of the telomerase catalytic unit (hTERT) in al., 1999; d’Adda di Fagagna et al., 1999; Samper et UT-7 and U937 human leukemia cell lines by using an al., 2000). In addition, telomere length is progressively IRES-e-GFP retrovirus. As expected, telomerase inacti- reduced with cell divisions (Blasco et al., 1997; Harley vation resulted in drastic telomere shortening, cytogenetic et al., 1990), due to the ‘end-replication problem’ instability and cell growth inhibition in all e-GFP positive (Hastie et al., 1990) and the putative exonuclease DN clones after 15 – 35 days of culture. However, despite activity in the CA-rich strand (Makarov et al., 1997). this initial response, 50% of e-GFP positive DN clones Telomere shortening in aging cells induces replicative with short telomeres escaped from crisis after 35 days of senescence (Morin, 1997). When telomeres reach a culture and recovered a proliferation rate similar to the critical size, chromosomes become unstable and under- control cells. This rescue was associated with a go end-to-end fusions, DNA fragmentation, and telomerase reactivation inducing telomere lengthening. mutations (Blasco et al., 1997; Gisselsson et al., We identified two pathways, one involving the loss of the 2001). Conversely, telomere length is stable in cells DN transgene expression and the other the transcrip- with long-lived replicative life spans, such as germ line tional up-regulation of endogenous hTERT with and embryonic cells, and in immortalized and tumor persistence of the DN transgene expression. Although cells (Wright et al., 1996). this second mechanism appears to be a very rare event Telomerase activation is the major mechanism that (one clone), these findings suggest that genomic instability maintains telomere integrity (Morin, 1989). The induced by short telomeres after telomerase inhibition human telomerase reverse transcriptase (hTERT) is might enhance the probability of activation or selection of an RNA-dependent DNA polymerase which synthe- telomere maintenance mechanisms dependent on hTERT sizes telomeric DNA using the human telomerase transcription. RNA (hTR) as a template (Colgin and Reddel, 1999; Oncogene (2002) 21, 8262 – 8271. doi:10.1038/sj.onc. Feng et al., 1995; Morin, 1989; Nugent and 1206054 Lundblad, 1998). Unlike germline and embryonic cells, somatic cells do not have telomerase activity, Keywords: telomere; telomerase; leukemia; retrovirus; with the exception of regenerative tissues such as cell death; therapy 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; Introduction Raymond et al., 1996). Enhanced telomerase activity represents an important step in the transformation Telomeres are dynamic DNA-protein complexes process of human cells, as the combined expression of (Blackburn, 2001) that cap the ends of linear 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 *Correspondence: A Bennaceur-Griscelli; E-mail: [email protected] activity may be associated with escape from senes- Received 3 July 2002; revised 13 September 2002; accepted 17 cence (Bodnar et al., 1998; Counter et al., 1998a; September 2002 Pendino et al., 2001). These findings validate Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8263 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 dominant- negative (DN) mutants (Hahn et al., 1999b; Zhang et Figure 1 Schematic representation of the Mig-R and Mig-R-DN al., 1999), or by G-quadruplex DNA ligands (Riou et constructs 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 per well. A dramatic decrease in telomerase activity telomerase regulation between human and murine cells was observed in 77% of U937 DN clones (31 out of (Sachsinger et al., 2001). Moreover, lessons learned 40) and 63% of UT-7 DN clones (47 out of 75), from knock-out mice have to be considered since compared to control vector clones (Figure 2a). The carcinogenesis is accelerated when both murine incidence of telomerase negative clones in DN clones telomerase RNA and p53 genes are inactivated (Chin was not different between clones with low and high et al., 1999). In addition, the possibility of resistance expression of e-GFP (data not shown). Consequences emerging from indirect telomerase-based therapies has of telomerase inhibition on telomere length were been recently observed in acute promyelocytic leukemia evaluated by Southern blot and Q-FISH in transduced cells treated by retinoic acid (Pendino et al., 2002). U937 and UT-7 clones after 25 – 35 and 20 – 25 PDs Thus it is possible that genetic instability induced by respectively (Figure 2b). Telomere length of the clones short telomeres might promote the selection of resistant transduced by the empty vector was not significantly sub-clones in human tumor cells subjected to a long different from uninfected parental cell lines (Figure 2b, term anti-telomerase strategy. As the possibility that lanes 1, 2, 3). During expansion, all DN U937 and UT- tumor cells might overcome telomerase inhibition has 7 clones exhibiting initially a reduced telomerase not been carefully evaluated in human tumors, we activity remained e-GFP positive and TRAP negative investigated this hypothesis in this work by studying (data not shown) indicating the stability of the the susceptibility of telomerase positive human leuke- transgene expression. Progressive telomere shortening mia cell lines to growth and death after short and long- was observed in all these clones, reaching values term telomerase inactivation by a DN-hTERT mutant around 2 kb (Figure 2b, lanes 4, 5, 6). Q-FISH (Hahn et al., 1999b). We studied the effects of analyses confirmed these data as a net decrease in telomerase inactivation in the myelomonocytic telomere fluorescence intensity was observed in DN U937 cell line (Harris and Ralph, 1985) and in the clones when compared to control clones (Figure 2c). UT-7 megakaryocytic cell line (Komatsu et al., 1991). We show that after an initial response defined by a DN affects cell growth and triggers cytogenetic instability critical telomere shortening, growth arrest and massive and cell death cell death, long-term culture results in the emergence of surviving clones, which escape from crisis by telomer- Clonogenicity (i.e. frequency of proliferating clones ase reactivation. Two mechanisms are responsible for from initial seeded cells) of DN cells was similar to this reactivation: the first one, occurring in both cell control cells in U937 and UT-7 cell lines (25 out of 60 lines, involves the loss of DN transgene expression control vector clones versus 51 out of 120 DN clones following telomere dysfunction and the second one, and 102 out of 180 control vector clones versus 154 out exclusively observed in one U937 DN clone, involves of 240 DN clones respectively), indicating the absence the increase in endogenous hTERT transcription with of cytotoxicity of DN in first mitoses. After 2 weeks of the persistence of the DN transgene expression. 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 Results telomeres in this cell line (5.5 kb compared to 3.5 kb in UT-7 cell line) was observed. The loss of viability DN inhibits telomerase activity and induces telomere induced by telomere shortening was confirmed by an shortening in leukemia cell lines increased rate of dead cells when analysing propidium U937 and UT-7 cells were transduced by the control iodide incorporation in both U937 and UT-7 DN vector Mig-R and Mig-R DN-hTERT retroviruses clones (Figure 3a). These clones displayed senescent (Figure 1). After three days of culture, e-GFP positive features such as enlarged cells and granular morphol- cells were sorted by flow cytometry and seeded at one ogy (data not shown). Since genetic instability is a cell per well. Telomerase activity was measured in direct consequence of shortened telomeres’ dysfunction, proliferating clones when they reached 1006103 cells we performed cytogenetic analyses. Additional end-to-

Oncogene Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8264

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: 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 compared to control vector clones. In the U937 cell chromosomes were observed in both U937 and UT-7 line, no sign of cytogenetic instability was found in DN clones with shortened telomeres (Figure 3b) when control vector clones (n=29 metaphases) whereas 36%

Oncogene Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8265 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 reversi- bility 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 Except the UT-7 DN14 and U937 DN95 clones, all other surviving DN clones became e-GFP negative, Figure 3 Cellular consequences of telomere shortening in U937 suggesting the loss of transgene expression (Figure 5a). and UT-7 clones at 25 – 35 and 20 – 25 PDs respectively after The DN transgene loss was confirmed by PCR in the e- transduction with DN or the control vector. (a) PI incorporation GFP negative clones (data not shown). The transgene/ histograms: the percentage indicates the cell fraction with sub-G1 DNA levels. (b) Representative metaphases of DN U937 (stan- IRES junction was analysed by RT – PCR in the clones dard coloration) and DN UT-7 (Banding G) clones harboring di- remaining e-GFP positive. In contrast to UT-7 DN14 centric or multicentric chromosomes. Arrows indicate the clone which had lost transgene expression despite the centromeres of dicentric and multicentric chromosomes 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 of DN clones’ metaphases (n=30) harbored dicentric, UT-7 DN14 clone without alteration of the IRES/GFP multicentric or ring chromosomes. Although uninfected sequence (data not shown), which may explain the UT-7 cell line and UT-7 control vector clones were persistence of the GFP expression. In order to found to exhibit a basal cytogenetic instability, we understand the increase in telomerase activity in observed a greater number of metaphases with U937 DN95 clone, we performed a semi-quantitative dicentric, multicentric or ring chromosomes in DN RT – PCR to calculate the relative quantity of clones compared to control vector clones (70% versus endogenous hTERT and DN mRNAs. The presence 20%, respectively) demonstrating an increase in genetic of the NruI site in the mutant cDNA discriminates the instability. endogenous hTERT from the mutant DN (Figure 6a). The relative percentages of endogenous and DN mRNAs were estimated and their respective quantities Reactivation of telomerase activity in DN clones at late were calculated after quantification of total endogen- passage leads to rescue from crisis ous and DN-hTERT transcripts using real-time In order to study the long-term efficacy of telomerase quantitative RT – PCR (Figure 6b). A significant inactivation in transduced DN e-GFP positive clones, threefold increase in hTERT transcription level we maintained slowly growing DN clones in culture (P=0.03, n=3) was observed in the U937 DN95 clone and monitored their outcome. Among these clones, 5 when compared to three control vector clones. As out of 11 U937 and 10 out of 18 UT-7 clones amplification of hTERT gene has recently been underwent massive cell death leading to complete described in human tumors (Zhang et al., 2000) we extinction. Interestingly, some DN clones continued analysed if an increase in hTERT gene copy number to survive despite an initially abolished telomerase had occurred in the U937 DN95 clone secondary to activity, very short telomeres and cytogenetic instabil- chromosomal instability. No hTERT amplification was ity. Indeed, 55% (6 out of 11 clones) of U937 and 44% observed as illustrated by FISH analysis (Figure 6c). (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 Discussion from crisis was attributed to telomerase reactivation in all residual survival DN e-GFP positive clones after the Several strategies to inhibit telomerase have been period of growth inhibition and cytogenetic instability reported in various tumor types but not in leukemias induced by critical telomere shortening. In contrast, a with successful in vitro and in vivo responses (Damm et

Oncogene Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8266

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 telomere size. This outcome is in agreement with those et al., 2001; Kondo et al., 1998; Riou et al., 2002; reported by others in solid tumors (Hahn et al., 1999b; Zhang et al., 1999). In this study, we analysed the short Herbert et al., 1999; Kim et al., 2001; Kondo et al., and long term consequences of an anti-telomerase 1998; Zhang et al., 1999). Growth inhibition is the strategy targeting hTERT in UT-7 and U937 human constant event observed in all studies, independent of leukemia cell lines. We show that the expression of the the mean of telomerase inhibition and the tumor type. DN-hTERT mutant was efficient with expected effects The delayed effect of telomerase inhibition depending such as telomere shortening inducing cell growth on initial telomere length suggests that telomerase inhibition, cell death, senescence and chromosomal inhibition has to be sufficiently long to allow tumor instability. However, this initial response was followed cells to reach critically shortened telomeres and the by the emergence of some clones, which escaped from expected crisis. Moreover, an efficient and permanent crisis in long-term cultures. The reversion of telomerase telomerase inhibition appears to be critical for the inhibition was due to the reactivation of telomerase by success of this approach regarding the potential risk of the loss of the transgene or by the up-regulation of additional genetic alterations induced by telomere endogenous hTERT mRNA transcription with contin- dysfunction (Gisselsson et al., 2001). ued expression of the transgene. The striking observation in our study was the Growth arrest and cell death, as cellular conse- appearance of long-term surviving clones overcoming quences of telomere shortening achieved by telomerase the telomerase inhibition. In both cell lines, 50% of the inhibition, were observed in both UT-7 and U937 clones, lacking initially telomerase activity, regained leukemia clones, after a delay depending on initial viability and returned to normal growth. Most of these

Oncogene Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8267

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. and to abrogate its DN function. It might be possible This event clearly occurred simultaneously with the that these chromosomal instability events depend on critical phase with very short telomeres and genetic the non-functional status of p53 in U937 cell line (Dou instability. The loss of the transgene was never et al., 1995). However, this mechanism of rescue observed in control clones nor in wild-type hTERT remains specifically dependent on the strategy used to transduced clones (data not shown) during the period inhibit telomerase and clearly limits the efficacy of a of expansion. Thus, this event may be considered as a gene therapy approach. In addition, this observation is consequence of the combination of the selection of potential interest as clones that had lost transgene pressure during long-term expansion and the telomere expression mimic an interruption of the telomerase dysfunction induced by the telomere shortening and not inhibition. A hypothesis which has to be confirmed by of the initial telomere-independent genomic instability, in vivo studies arises from these observations: an especially in UT-7 cell line. Genomic alterations, such inadequate arrest of telomerase inhibition in the period as DNA fragmentations and mutations (Gisselsson et of chromosomal instability might select clones with a al., 2001), induced at late passage by telomere phenotype distinct from initial tumor cells due to dysfunction could contribute to alter the transgene additional genomic disorders.

Oncogene Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8268

Figure 6 Overexpression of hTERT in U937 DN95 clone. (a) Semi-quantitative RT – PCR encompassing reverse transcriptase re- gion 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 the hTERT mutant was still altered at the DNA level, clone (U937 DN95) which rescued from crisis by a we can suppose that the DN protein was still drastic reactivation of telomerase despite the stable functional. In contrast to a recent work in HEK 293 expression of DN transgene. Since the catalytic site of cells (Zhang et al., 1999), we found an increase in

Oncogene Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8269 endogenous hTERT mRNA. A similar mechanism was France) and 10 ng/ml GM-CSF (Novartis, Nanterre, France) also recently reported in murine RenCa clones for the UT-7 cell line. subjected to a mTERT mutant (Sachsinger et al., 2001). Whether this mechanism depends on a TERT Plasmids component targeting strategy or may occur in alter- native modes of telomerase inhibition remains to be The Murine Stem Cell Virus retroviral vector Mig-R1 explored. Nevertheless, the increase in endogenous containing encephalomyocarditis virus (ECMV) internal hTERT sub-units may have overcome the dominant ribosomal entry sequence (IRES) and green fluorescent protein (e-GFP), was kindly provided by Warren S Pear negative effect in accordance with the putative (University of Pennsylvania, Philadelphia, PA, USA). Mig- mechanism of telomerase inactivation by the DN- R2 plasmid was constructed by replacing ECMV-IRES by hTERT mutant (Arai et al., 2002). When comparing Vascular Endothelial Growth Factor IRES. Mig-R-DN hTR expression in the U937 DN95 clone and the U937 vectors were constructed by subcloning the cDNA sequence DN12 clone, which had lost the transgene expression, of the DN-hTERT mutant from pBABE-puro-DN plasmid with the control clones, we did not observe any provided by Robert A Weinberg (MIT, Cambridge, MA, significant difference (data not shown), which is USA) (Hahn et al., 1999b) into either Mig-R1 or Mig-R2 consistent with the fact that hTERT is the limiting (Figure 1). component for telomerase activity (Counter et al., 1998b). It is now well established that c-Myc or Sp1 Transfections and infections are involved in the up-regulation of hTERT at the transcription level (DePinho et al., 1991; Kyo et al., Retroviral supernatants of control and DN vectors were 2000). However, RT – PCR and Western blot analyses prepared as previously described (Pendino et al., 2001) for further infections of U937 and UT-7 cell lines in the presence of c-Myc and Sp1 expression in the U937 DN95 clone of 4 mg/ml of Polybrene (Sigma-Aldrich, Saint Quentin revealed no difference with the control vector clones Fallavier, France). E-GFP positive cells were sorted 2 days (data not shown). Furthermore, we did not find any later by flow cytometry (FACSvantage, Becton Dickinson, Le hTERT gene amplification as previously described in Pont de Claix, France), according to a low or a high intensity various tumor cells (Zhang et al., 2000). Nevertheless of fluorescence, and seeded at one cell per well in a 96-well one can speculate that molecular events leading to plate. The constant expression of e-GFP was controlled twice endogenous telomerase over-expression have been a week by flow cytometry during all the period of cell either induced or selected by telomerase inhibition expansion. consequences since such an up-regulation did not occur in control vector clones. This finding suggests that anti- Telomerase activity assays telomerase strategy, targeting hTERT, could select rare variant sub-clones enhancing endogenous telomerase to Telomerase activity was measured by a modified TRAP assay, using TRAPeze kit (Intergen, Oxford, UK). Semi by-pass the crisis. Although this kind of rescue seems quantitative measurement of telomerase activity was to be a rare event, we cannot formally exclude that performed after polyacrylamide gel electrophoresis using the other clones were able to rapidly evade the inhibitory Storm 840 instrument (Amersham Lifescience, Bondoufle, mechanism, as telomerase inactivation in output e- France). Real-time quantitative TRAP using ABI PRISM GFP-positive clones was partially observed in 60 – 80% 7700 Detection System (Perkin Elmer Applied Biosystems) of input clones. was performed as described (Hou et al., 2001). The standard In conclusion, we show the potential interest of the curve was established using serial dilutions of HL60 protein anti-telomerase strategy in human leukemia. Moreover, extract in CHAPS buffer. One arbitrary unit (a.u) was we demonstrate the possible emergence of human defined as the telomerase activity of 1 ng of HL60 protein leukemia cells overcoming telomerase inhibition by a extract. transcriptional up-regulation of endogenous hTERT. Thus, the appearance of variant clones has to be taken Telomere length analysis into account for the strategy based on telomerase Genomic DNA was digested with 10 units each of HinfI and inhibition. The combination of two telomerase inhibi- RsaI (Roche, Meylan, France), underwent a 0.7% agarose gel tors targeting distinct elements of telomerase complex electrophoresis and was transferred onto a nylon membrane or its substrate (Riou et al., 2002) and the association for further hybridization to a digoxigenin-labeled probe with synergistic therapies such as radiotherapy (Goyti- (TTAGGG)3. Revelation of TRF was performed by solo et al., 2000; Wong et al., 2000) or chemotherapy chemiluminescence detection. Telomere length was also (Lee et al., 2001) may contribute to limit the emergence evaluated by Q-FISH using a Cy3 labeled PNA probe of such resistant sub-clones. (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 Materials and methods fluorescence microscope. CCD camera captured nuclei were analysed using Smart Capture Software (Vysis, Voisins le Cell lines Bretonneux, France) and quantitative analysis of image was The U937 myelomonocytic and UT-7 megakaryocyte human performed with Quips IP LAB software. Average results of leukemia cell lines were cultured in alpha MEM medium telomeric fluorescence were expressed as fluorescence intensity supplemented with 10% FCS (Invitrogen, Cergy-Pontoise, arbitrary units.

Oncogene Telomerase inhibition in human leukemia cell lines F Delhommeau et al 8270 TCTCCCCAAGCCGT-3’), and b2-microglobulin (b2-M) Cellular assays housekeeping gene as external standard (5’-TCCTGAAGCT- Cell growth and viability were determined using trypan blue. GACAGCATTCG-3’ and 5’-TCCTAGGAGCTACCTGT- DNA content was analysed by flow cytometry using 25 mg/ml GGAG-3’). Total RNA was extracted by RNA-B (Q-Biogen, propidium iodide (PI) (Sigma) in 0.1% NP40 citrate buffer Illkirch, France) and reverse transcribed using MuLV reverse with 0.5 mg/ml of RNAse A (Boehringer). transcriptase (Perkin Elmer Applied Biosystems). To discri- minate endogenous hTERT from DN, RT – PCR products were digested by NruI (New England Biolabs, MA, USA). Cytogenetic analysis and in situ hybridization study of hTERT Real-time quantification of total unspliced hTERT transcripts gene copy number was performed using the LightCycler TeloTAGGG hTERT Standard cytogenetic techniques were used for chromosome quantification kit (Roche). Serial dilutions of Mig-R1-hTERT preparations. Metaphases were analysed in giemsa and G or plasmid were used as standards. 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 Acknowledgments studied by FISH. The BAC clone 518C13 was kindly We are grateful to S Stewart and RA Weinberg and to WS provided by S Burns (Beatson Laboratories, University of Pear for providing the constructs, to S Burns for providing Glasgow, Glasgow, UK). Briefly, the probe was prepared by the BAC clone and to F Forestier for the use of the labeling the BAC DNA with digoxigenin using Dig Nick LightCycler. We thank H Raslova and P Foliot for FISH translation Kit (Roche) and hybridization was performed as techniques and J Dando for critically reading the manu- previously described (Bryce et al., 2000). script. 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 RT – PCR analyses contre le Cancer (ARC no 5606 and 7364), from Institut The expression of endogenous and exogenous hTERT Federatif de Recherche of Institut Gustave Roussy (IFR mRNAs was analysed by RT – PCR with primers specific 2000), from the Fondation de France (no 98001281) and for hTERT (5’-CTGTCGGAAGCAGAGGTCAG-3’ and 5’- from Universite´ Paris-Sud (BQR 2000 no CR112). F CTCCATGTCGCCGTAGCACA-3’), hTERT/IRES junction Delhommeau was supported by INSERM and A Thierry (5’-ATCCTCCTGCTGCAGGCGTA-3’ and 5’-GAGCAA- by the Ligue contre le Cancer.

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