Telomerase inhibition in a mouse cell line with long telomeres leads to rapid telomerase reactivation

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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

w w w. e l s e v i e r. c o m / l o c a t e / y e x c r

Research Article

Telomerase inhibition in a mouse cell line with long telomeres leads to rapid telomerase reactivation Delphine T. Marie-Egyptienne a,b , Marie Eve Brault a,b , Shusen Zhu b , Chantal Autexier a,b,c,⁎ a

Department of Anatomy & Cell Biology, McGill University, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec, Canada H3T 1E2 b Bloomfield Centre for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montréal, Québec, Canada H3T 1E2 c Department of Medicine, McGill University, Montréal, Québec, Canada H3A 2B2

ARTICLE INFORMATION

ABS T R AC T

Article Chronology:

The indefinite growth of cancer cells requires telomere maintenance, which, in the majority of

Received 12 July 2007

mammalian cancers is mediated via the enzyme telomerase. The core components of

Revised version received

telomerase are a catalytic reverse transcriptase (hTERT in human, mTERT in mouse) and an

16 September 2007

RNA (TR) that contains the template for the replenishment of telomeres. Fundamental

Accepted 26 October 2007

differences in human and mouse telomerase and telomere biology should be considered when

Available online 7 November 2007

using mouse models for the study of human cancers. The responses to telomerase inhibition by the expression of a catalytically-inactive dominant-negative mutant of hTERT (hTERT-DN)

Keywords:

vary in human cells with different telomere lengths. Only one similar study has been

Telomerase inhibition

performed in a mouse cell line with short telomeres (RenCa, 7 kb). Thus, we asked whether the

Telomerase reactivation

responses to telomerase inhibition are also telomere-length dependent in mouse cells by

Mouse cell line

analyzing long-term stable expression of mTERT-DN in the CB17 cell line (telomere length,

Long telomeres

11 kb). A brief initial telomerase inhibition was insufficient to mediate telomere shortening and

Dominant-negative TERT

led to extremely rapid telomerase reactivation due to an increase in the level of expression of the endogenous mTERT. Thus, mouse cells, in contrast to human cells may not tolerate telomerase inhibition by introduction of mTERT-DN, independently of telomere length. © 2007 Elsevier Inc. All rights reserved.

Introduction Telomeres are nucleoprotein structures consisting of tandem repeats of short G-rich sequences that cap the ends of chromosomes and maintain their stability [1]. Human telomeres span 2 to 15 kilobase pairs (kb), whereas in Mus musculus, they extend from 10 to 60 kb [2]. Telomeres are synthesized by a telomere-specific DNA polymerase, telomerase. The enzyme's core components are a specialised catalytic reverse transcriptase subunit (hTERT in human, mTERT in mouse) and an RNA subunit (hTR in human,

mTR in mouse) that contains the template for the replenishment of the telomeric repeats [3]. Because telomeres act as a molecular clock that regulates the proliferative limit of human cells, telomere maintenance and telomerase are crucial for the continued growth of normal and malignant cells [4,5]. Telomerase is expressed in cells of highly proliferative tissues [4], allowing them to divide extensively. However, because most human somatic cells lack telomerase, they eventually reach a non-dividing state termed replicative senescence [5]. The lifespan of human

⁎ Corresponding author. 3755 Côte-Sainte-Catherine, Lady Davis Institute for Medical Research, Montréal, Québec, Canada H3T 1E2. Fax: +1 514 340 8295. E-mail address: [email protected] (C. Autexier). 0014-4827/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.10.020

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somatic cells can be increased by inactivating both p53 and pRb [5], but their telomeres continue to shorten. The cells then reach crisis where the vast majority dies [5]. Cells that survive crisis are immortal and often cancerous [5,6]. Forced hTERT expression in pre-senescent cells restores telomerase activity and maintains telomeres, resulting in an extension of cellular lifespan [7]. Indeed, the indefinite growth of 85–90% of human tumors has been found to parallel telomerase activity [4,6]. In contrast to most normal human tissues, many adult mouse tissues possess active telomerase [8]. The presence of telomerase in normal mouse somatic cells certainly plays a role in their spontaneous immortalization, for which the inactivation of the p53 pathway is sufficient [9]. Also, cultured mouse cells undergo growth arrest prior to significant telomere erosion indicating that mouse senescence is not telomere-based [10,11]. Similar to human tumors, mouse tumors express elevated levels of telomerase activity [12,13]. Furthermore, the abrogation of telomerase activity in mouse cancerous cells impairs their metastatic potential [14] underlying the importance of telomerase in tumor formation and progression. Many anti-cancer strategies targeting telomerase in human cells have been successful in vitro [15]. Telomerase inhibition leads to telomere shortening, decreased cell viability and in most cases cell death by apoptosis [16–21]. hTERT-DN is a catalytically-inactive dominant-negative hTERT mutant that confers loss-of-function to endogenous human telomerase and leads to telomere shortening in telomerase-positive human cells. The limitation of telomerase inhibition in cancer cells is the lag phase associated with the time required for telomeres to shorten sufficiently to observe anti-proliferative effects. A consequence of this lag phase is that hTERT-DN expression results in apoptosis of human cells with short (3–7 kb) but not long telomeres (10–12 kb) [16– 18,21,22]. Human cells with long telomeres exhibit an initial reduction of telomerase activity and limited telomere shortening that is reversed due to loss of hTERT-DN expression, transcriptional upregulation of endogenous hTERT or by an unknown mechanism [16,21]. Strikingly, expression of TERTDN elicits different responses in mouse and human tumor cell lines with short telomeres. In a renal mouse cancerous cell line, RenCa that harbours telomeres 7 kb in length, the expression of mTERT-DN results in only transient telomerase inhibition, telomere shortening and loss of cell viability [23]. Similarly to human cells with long telomeres, the RenCa cells expressing mTERT-DN transcriptionally upregulate TERT expression, despite the persistence of TERT-DN expression. Thus, the responses to telomerase inhibition may be regulated differently in human and mouse immortal cell lines [23]. Because the responses to telomerase inhibition by hTERT-DN vary in cells with different telomere lengths, we asked whether the responses to telomerase inhibition are also telomere-length dependent in mouse cell lines. We analyzed the consequences of telomerase inhibition by the expression of mTERT-DN in a mouse cell line with telomeres greater than 7 kb in length. In the CB17 cell line, whose telomeres are 11 kb, the brief initial telomerase inhibition resulting from the expression of mTERT-DN was followed by rapid telomerase reactivation due to an increase in the level of expression of the endogenous mTERT. Therefore, it seems that independent of telomere length, mouse cells may not tolerate

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telomerase inhibition by introduction of a dominant-negative mTERT. This observation reinforces the inherent differences between human and mouse telomerase regulation in terms of cellular proliferation.

Materials and methods Constructs p-HA-mTERT-WT and p-HA-mTERT-DN were generated from the pcDNA3.1 HA mTP2 plasmid [24]. The mTERT-DN cDNA was made from pcDNA3.1 HA mTP2 by site-directed mutagenesis, based on the Quick Change Site-Directed Mutagenesis method (Stratagene). Primers used were: 5′mTERT-DN (5′CTTTTACGTTTTGTTAACGACTTTCTGTTG-3′) and 3′mTERTDN (5′-CAACAGAAAGTCGTTAACAAAACGTAAAAG-3′). Both BamHI–NotI treated mTERT-WT and mTERT-DN cDNAs were then sub-cloned into the BamHI–NotI treated pcDNA3-neomycin plasmid (Invitrogen) (from Anne Gatignol, Lady Davis Institute, Montreal, Canada). The constructs phTERT-WT and phTERT-DN were previously described [25].

Cell lines, transfection and cellular extracts The LY-S cell line, a mouse lymphoma cell line (ATCC) (provided by Michael Pollack, Lady Davis Institute, Montreal, Canada) [26] was cultured in Fischer's media (MP Biomedicals) with 10% FBS supplemented with 7.5% sodium bicarbonate and sodium pyruvate. The CB17 cell line (gift from Predrag Slijepcevic, Brunel Institute of Cancer Genetics and Pharmacogenomics, Uxbridge, United Kingdom) [27] was grown in Waymouth media with 10% FBS. Stable cell lines were obtained by Lipofectamine 2000 (Invitrogen) transfection with the vector pcDNA3 alone, p-mTERT-WT or p-mTERTDN. Clonal populations were selected with 600 μg/ml neomycin for 3–4 weeks, and were routinely subcultured under selection at a 1:4 split ratio as they reached confluence. Whole cell protein extracts were made as previously described [28].

RNA isolation and RT-PCR Total RNA was isolated using TRIzol (Invitrogen). Reverse transcription of the RNAs was followed by PCR to detect the ectopically expressed mTERT (mTERT-WT or mTERT-DN) with a primer specific for the HA tag at the 5′ of mTERT. The HA-mTERT-WT cDNA was detected with the primers 5′HA-mTERT, 5′-TACCCATACGATGTTCCTGACTATGC-3′ and 3′mTERT, 5′-GGAATGTGCTCTCTGTGATGTAAAAG-3′. The HA-mTERT-DN cDNA was detected with a nested PCR; the first PCR step was performed with the 5′ and 3′mTERT primers described above, and the second PCR step was performed with the 5′HA-mTERT and the mTERT513R 5′-ACTCGGCTCAACAGTAGCATC-3′ primers. The endogenous mTERT mRNA was detected with the primer mTERT endogenous 5′ (5′-GGTTGAAGTGTCACGGTC3′) and a primer specific to the 3′ untranslated region of mTERT, mTERT endogenous 3′ (5′-GTTCATCTAGCGGAAGGAG-3′). Mouse GADPH was amplified with primers RT11 and RT12 as previously described [28].

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are shared with other reverse transcriptases [29]. In particular, motif C contains three conserved aspartic acids essential for the activity of the enzyme [29]. To inhibit the endogenous enzyme, we first constructed a point mutant of mTERT, mTERT-DN. We substituted the aspartic acid at position 861 in mTERT (equivalent to 868 in hTERT) by an asparagine residue (D861 → N861) [16,23]. We verified that the engineered mTERT-DN mutant effectively inhibits telomerase activity in vitro. As mTERT and mTR weakly reconstitute enzyme activity in vitro [30,31] (Fakhoury,

Fig. 1 – (A) Upper panel. Telomerase complexes were reconstituted in RRL, and telomerase activity was detected using TRAP. IC, internal PCR control. Lower panel. In vitro synthesis of 35S-methionine-labeled hTERT and mTERT (WT and DN). 1 μl of the corresponding RRL reaction was analyzed by SDS-PAGE. (B) 1000, 100 and 70 ng of proteins from whole cell extracts of the parental cell line (CB17p) were tested for telomerase activity by TRAP assay.

In vitro reconstitution of telomerase and TRAP assays In vitro reconstitution of telomerase in rabbit reticulocyte lysates (RRL), TRAP assays and its quantifications were previously described [28].

Telomere length analysis Telomere length was determined by Telomeric Restriction Fragment (TRF) analysis as previously described [50].

Results Telomerase reconstituted with mTERT containing a point mutation in a conserved reverse transcriptase motif C aspartic acid residue is catalytically inactive in vitro The RT domain of the TERT subunit is essential to mediate catalysis via the seven RT motifs (1 and 2, A, B′, C, D and E) that Fig. 2 – (A) Expression profiles of ectopic mTERT in the clones at PD0. CB17 clones stably transfected with p-HA-mTERT or p-HA-mTERT-DN were analyzed for the expression of ectopic mTERT by RT-PCR. GAPDH was used as a control for the integrity of the cDNAs and the p-HA-mTERT-WT plasmid was used as a positive control for the HA-specific PCR. (B) Telomerase activities of the selected clones detected by TRAP assay. 80 and 20 ng of whole cell protein extracts from the CB17p cells, mTERT-DN- and mTERT-WT-expressing clones were tested for telomerase activity. IC, internal PCR control. %WT, average percentage of WT telomerase activity derived by quantification of the CB-mDN clones' telomerase activities relative to the WT telomerase activity (n ≥ 2). The brackets denote the presence of PCR primer dimers.

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J. Marie-Egyptienne, D.T. and Autexier, C., unpublished), we reconstituted the telomerase enzymes by expressing TERTs in the presence of hTR using an in vitro RRL system and analyzed their activities by TRAP assay. mTERT and hTR reconstituted telomerase enzyme exhibited slightly less activity compared to the hTERT and hTR reconstituted telomerase enzyme (Fig. 1A, lanes 1 and 3) [32]. Despite equivalent synthesis of both hTERT and hTERTDN, or mTERT and mTERT-DN proteins, enzymes reconstituted with either hTERT-DN [25] or mTERT-DN were inactive (Fig. 1A, upper and lower panels, lanes 2 and 4).

CB17 is a telomerase-positive mouse cell line with telomeres 11 kb in length At present, RenCa is the only mouse cell line in which mTERTDN expression has been analyzed; this cell line is characterized by short telomeres 7 kb in length [23]. To test whether the response to telomerase inhibition is telomere-length dependent in mouse cells, we selected a mouse telomerase-positive cell line, CB17, with telomeres longer than 7 kb. We confirmed that CB17 cells exhibit detectable telomerase activity by TRAP assay (Fig. 1B) [26]. For telomere length analysis, we included the LY-S mouse lymphoma cell line as a marker for short telomeres. The telomere lengths of CB17 and LY-S have been previously reported to be 11 and 7 kb by Q-FISH analysis [26]. We quantified the telomere length of the CB17 at 11 kb, which is longer than the calculated telomere length of the LY-S at 5 kb (Fig. 3A, lanes 1 and 2). We subsequently stably expressed mTERT-DN in the CB17 cells to test the effects of the long-term expression of mTERT-DN on the viability of a mouse cell line with long telomeres.

Effect of mTERT-DN expression on telomerase activity of CB17 cells In human or mouse cells, the presence of hTERT-DN or mTERTDN respectively leads to inhibition of telomerase activity [16– 18,21,23]. We first assessed by RT-PCR the expression of ectopic HA-mTERT in the mTERT-WT and mTERT-DN-transfected clones at the time of isolation (Fig. 2A). We chose one mTERTWT-transfected clone, CB-mWT2 and two mTERT-DN-transfected clones, CB-mDN4 and CB-mDN7 that expressed the ectopic HA-mTERT-WT or HA-mTERT-DN (Fig. 2A, upper and lower panels, respectively). We then tested if the expression of mTERT-DN could have a dominant-negative effect on the endogenous telomerase enzyme by measuring telomerase activity of cell extracts from the parental cell line, the mTERT-DN- and mTERT-WTexpressing clones by TRAP assay (Fig. 2B). The cell extracts were prepared immediately upon isolation of the clones (population doubling 0: PD0). The mTERT-WT-expressing clone, CB-mWT2, exhibited levels of telomerase activity comparable to those of the parental cell line (Fig. 2B, upper panel). Both CB-mDN4 and CB-mDN7 demonstrated reduced telomerase activities compared to the parental cell line (Fig. 2B, lower panel). Quantification of telomerase activity (n N 2 using 80 ng protein) revealed that endogenous telomerase activity was inhibited by 83% and 66% in the CB-mDN4 and CB-mDN7 clones, respectively (bottom of Fig. 2B).

Fig. 3 – (A) TRF analysis of the CB-mDN clones at PD0 and PD18. (B) Growth curves of the CB-mWT2, CB-mDN4 and CB-mDN7 clones indicating culture growth (PD) as a function of days.

Effects of mTERT-DN expression on telomere length and cell proliferation of CB17 cells In human and mouse cell lines, the inhibition of telomerase by TERT-DN expression leads to initial telomere shortening [16– 18,21]. Hence, we tested the consequences of mTERT-DN expression on telomere maintenance by performing TRF analysis on genomic DNA isolated from CB17p cells, CB-mDN4 and CBmDN7 clones at early (PD2) and late population doublings (PD18). The telomeres of the CB-mDN4, CB-mDN7 and CB-mWT2 clones were maintained at 11 kb between PD2 and PD18, similar to the telomere lengths of the parental cell line (Fig. 3A, lanes 3 to 6).

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Extensive telomere shortening commonly correlates with decreased cell growth and proliferation [5,16,17]. We monitored the growth of the CB-mWT2, CB-mDN4 and CB-mDN7 clones with increasing population doublings (Fig. 3B). The growth of the CB-mDN clones was similar to that of the CBmWT2 clone. The lack of telomere shortening thus correlated with a lack of decreased cell growth and proliferation.

Rapid reactivation of telomerase activity in the CB-mDN4 and CB-mDN7 clones The introduction of hTERT-DN in human cells with long telomeres leads to initial telomerase inhibition and telomere shortening, but reactivation of telomerase activity and telomere maintenance or elongation at later population doublings [16,21]. A similar phenotype is observed in the RenCa mouse tumor cell line with short telomeres [23]. We tested whether telomerase reactivation had occurred in the CB-mDN clones, which could explain the lack of telomere shortening and decreased cell proliferation.

We performed TRAP assays on cell extracts isolated from the CB-mDN clones at PD2, PD4, and PD12. Extracts from the parental cell line and from the clones at PD0 were included for comparison. Both CB-mDN clones reactivated telomerase activity to levels comparable or greater than those of the parental cell line (Fig. 4A). Reactivation of telomerase activity was observed rapidly by PD2 indicating that the telomerase inhibition observed at the time of clonal isolation (PD0) had been lost. Thus the absence of telomere shortening with increasing population doublings of the CB-mDN clones was most likely due to the extremely rapid reactivation of telomerase activity. Human cells with long telomeres that express hTERT-DN reactivate telomerase at late population doublings due to either loss of hTERT-DN expression, transcriptional upregulation of endogenous hTERT, or by an unknown mechanism [16,21]. In the mouse RenCa cell line with short telomeres, telomerase activity is similarly reactivated due to upregulation of endogenous mTERT despite the persistence of mTERT-DN [23]. To understand the mechanism of telomerase reactivation

Fig. 4 – (A) Telomerase activities of the selected clones at increasing PDs detected by TRAP assays. IC, internal PCR control. The brackets denote the presence of PCR primer dimers. (B) Expression profiles of ectopic and endogenous mTERT in the CB-mDN4 and CB-mDN7 clones at serial PDs. GAPDH was used as a control for the integrity of the cDNAs and the p-HA-mTERT-WT plasmid was used as a positive control for the HA-specific PCR.

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in the CB-mDN clones, we assessed ectopic and endogenous mTERT expression by RT-PCR. HA-mTERT-DN expression persisted in the CB-mDN stable clones with increasing population doublings (Fig. 4B, upper panel). Low levels of endogenous mTERT in the CB17p cells were detected (Fig. 4B, lower panel). Interestingly, despite the inhibition of telomerase activity in the CB-mDN clones at PD0, low levels of endogenous mTERT were detectable. However, upregulation of endogenous mTERT expression in the CB-mDN4 and CB-mDN7 clones was readily apparent by PD2 and was maintained to PD12 (Fig. 4B, lower panel). The higher level of endogenous mTERT expression in the mTERT-DN clones than in the parental cell line has previously been observed in the RenCa cell line [23]. Although other unidentified mechanisms might also contribute to the reactivation of telomerase activity, we concluded that the reactivation observed in the stable CB-mDN clones was most probably due to the rapid upregulation of the endogenous mTERT, despite persistent expression of the mTERT-DN transgene.

Discussion Our results in the CB17 cell line contrast sharply from those obtained in human cell lines with similarly long (10–12 kb) telomeres treated with hTERT-DN. In these studies, marked telomere shortening occurred due to a longer duration of telomerase inhibition prior to telomerase reactivation [16,21]. While telomerase inhibition in the mouse RenCa cell line led to some telomere shortening, both mouse RenCa cells with short telomeres and CB17 cells with longer telomeres may not tolerate the inhibition of telomerase by mTERT-DN [23]. Thus, in contrast to the telomere-length-dependent response of human cells to telomerase inhibition by hTERT-DN, mouse cells seemingly react in a telomere-length-independent fashion to telomerase inhibition by expression of mTERT-DN. Telomerase reactivation is extremely rapid in our mTERTDN-expressing clones. Though equivalent numbers of cells were plated, transfections with the mTERT-DN plasmid generated fewer clones compared to transfections with the vector or the mTERT-WT plasmid. Moreover, many of the isolated clones did not display telomerase inhibition, despite expression of the mTERT-DN (data not shown). Thus, it is possible that these isolated clones counteracted telomerase inhibition, probably by upregulating the endogenous TERT subunit. The lack of observable telomere shortening in the CBmDN clones we analyzed is likely due to the short period of telomerase inhibition. However, we cannot exclude that telomere shortening did not occur during selection if, as a result of clonal variation, the stable clones we chose initially had longer telomeres than the parental cells [16]. A telomere lengthening-independent role of telomerase may prevent the long-term loss of enzyme activity in the CB17 cell line. The importance of telomerase for viability and proliferation of both human and mouse cells is not only due to its ability to generate telomeric repeats de novo [33]. Telomerase overexpression in dividing and differentiated human and mouse cells provides protection against diverse apoptotic insults [34–36] and telomerase can be upregulated in cells upon treatment with apoptosis-inducing stimuli [37]. Telo-

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merase overexpression in transgenic mice with long telomeres leads to increased wound healing upon mutagenic insults and increased rate of neoplasia [38–40]. Thus, it appears that telomerase can promote proliferation of cells with long telomeres. In human mammary epithelial cells, telomerase overexpression bypasses the need of exogenous mitogens for cell growth and leads to the upregulation of genes that control growth [41]. Some of the effects of telomerase described above appear to be independent of catalytic activity and/or the telomerase RNA component [42,43]. Since the CB17 cell line appears not to tolerate the inhibition of telomerase activity, it is possible that the activity is required for proliferation, independent of telomere addition. Our data and those obtained in the RenCa cell line suggest that mouse cells may not tolerate telomerase inhibition by mTERT-DN. Although not many anti-telomerase strategies have been exploited in mouse cells, it appears that the intolerance to telomerase inhibition may be TERT-DN-specific. Indeed, mouse telomerase inhibition by other strategies leads to cell death [14,23,44]. For example, anti-mTR ribozyme-treated murine melanoma cells experience a decrease in mTR expression and telomerase activity, telomere shortening and increased rate of apoptosis [14]. These strategies, unlike TERTDN, do not lead to the reactivation of the endogenous enzyme [14,44]. Mice have been extensively used as models for human cancers [45]. In this regard, the mouse telomerase models have contributed to the understanding of the role of telomere maintenance in human cancer [46–49]. Nevertheless, the data obtained from the use of TERT-DN to inhibit telomerase in human or mouse cells illustrate the differences in the response to telomerase inhibition between mouse and human cells. The rapid reactivation and lack of telomere shortening upon treatment with TERT-DN have not been reported in human cells. A better comprehension of the differences and similarities between human and mouse telomere and telomerase biology may allow more accurate interpretation of cancer biology findings derived using mouse models.

Acknowledgments We thank Drs L. Harrington, P. Slijepcevic, A. Gatignol, and M. Pollack for the constructs and cell lines. We thank Graeme Nimmo, Johans Fakhoury for helpful discussion and critical reading of the manuscript and J. Demers for help in the generation of the p-mTERT constructs. This work was funded by a Canadian Institute for Health Research Grant MOP68844 to C. Autexier. C. Autexier is a chercheur-boursier of the Fonds de la Recherche en Santé du Québec. D.T. Marie-Egyptienne is the recipient of a McGill University Graduate Student Fellowship and M.E. Brault is a Cole Foundation Fellow.

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