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Tumor Biology

Telomerase Inhibition in RenCa, a Murine Tumor Cell Line with Short Telomeres, by Overexpression of a Dominant Negative mTERT Mutant, Reveals Fundamental Differences in Telomerase Regulation between Human and Murine Cells

Jana Sachsinger, Eva González-Suárez, Enrique Samper, Rüdiger Heicappell, Markus Müller and María. A. Blasco
Jana Sachsinger
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Eva González-Suárez
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Enrique Samper
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Rüdiger Heicappell
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Markus Müller
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María. A. Blasco
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DOI:  Published July 2001
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Abstract

In contrast to human primary fibroblasts, mouse embryonic fibroblasts have telomerase activity, immortalize spontaneously in culture, and can be neoplastically transformed by oncogenic insult. Ectopic expression of the human telomerase catalytic subunit, human telomerase reverse transcriptase (hTERT), in human primary cells allows both spontaneous immortalization and neoplastic transformation by oncogenes. This suggests that telomerase activity, as well as the fact that mouse telomeres are longer than human telomeres, may explain some of the differences in cellular control between human and murine cells. Telomerase inhibition in immortal or transformed human cells using dominant negative hTERT mutants leads to telomere shortening and cell death. Here we study the effect of expression of a dominant negative mutant of the catalytic subunit of mouse telomerase, mTERT-DN, in a murine kidney tumor cell line, RenCa, whose telomeres are similar in length to human telomeres. After showing initial telomerase activity inhibition and telomere shortening, all clones expressing mTERT-DN reactivated telomerase and showed normal viability, in contrast with that described for human cells. This efficient telomerase reactivation coincided with a significant increase in the endogenous TERT mRNA levels in the presence of mTERT-DN expression. The results presented here reveal the existence of fundamental differences in telomerase regulation between mice and man.

INTRODUCTION

Telomerase is the cellular DNA polymerase involved in telomere replication (1) . Telomeres are the ends of eukaryotic chromosomes and, in vertebrates, consist of tandem DNA repeats of the TTAGGG sequence and of associated proteins (2 , 3) . Telomeres are proposed to protect chromosome ends from degradation, recombination, and DNA repair activities. Loss of telomeric function, either by the loss of telomeric sequences or the mutation of telomere binding proteins, is associated with increased chromosomal instability and the loss of cell viability (4, 5, 6, 7, 8, 9, 10) . Telomerase is a specialized reverse transcriptase that synthesizes telomeric repeats onto chromosome ends (11) . The telomerase enzyme is composed of a catalytic protein subunit known as TERT3 (8 , 12, 13, 14, 15, 16, 17, 18) , an RNA molecule or Terc, which is used as template for the addition of new telomeric repeats (19, 20, 21, 22, 23) , and associated proteins (24, 25, 26, 27, 28, 29) . The introduction of a constitutively expressed human telomerase catalytic subunit, hTERT, into human cells with a limited life span is sufficient to stabilize their telomeres and, in some cases, to extend their life span indefinitely without inducing changes associated with transformation (30, 31, 32, 33, 34) , supporting the idea that telomere maintenance by telomerase is essential for immortal growth. Telomerase can also cooperate with oncogenes to transform primary human cells in culture, suggesting that telomere maintenance by telomerase contributes to malignant transformation (35) . These findings led to the proposal that telomerase inhibition in human cancers would provoke telomere shortening, and compromise tumor growth with time. Indeed, telomerase inhibition in human tumor cell lines using dominant negative versions of hTERT resulted in telomere shortening and cell death (36 , 37) , suggesting that telomerase inhibition may be an effective way to halt tumor growth (38) . RenCa is a murine renal carcinoma cell line (39) whose telomeres are similar in length to those of human immortal cells lines. Using this cell line, we have studied the effects of expressing a dominant negative Mt of mTERT in murine tumor cells. The results presented here reveal the existence of fundamental differences in telomerase regulation between mice and man.

MATERIALS AND METHODS

Plasmids, Cell Culture, and Transfection

The mouse RenCa renal carcinoma cell line, all pBabe- and pDN-transduced clones, as well as the tumor-derived cell lines were cultured in DMEM supplemented with 10% FCS. Site-directed mutagenesis of TERT residues Asp (861) and Asp (862) to Ala was carried out using the Sculptor in vitro mutagenesis system (Amersham Pharmacia Biotech, Stockholm, Sweden) using the full-length mouse TERT cDNA previously cloned into Bluescript SK as template. 4 An EcoRI fragment containing the full-length Mt TERT cDNA and 3′-untranslated sequences was subcloned into a pBabe retroviral vector harboring a puromycin resistance gene.

Retroviral constructs were introduced by standard calcium phosphate transfection into Phoenix packaging cells, and the viral supernatant was used to transduce RenCa cells as described (5) . Transduced clones were selected with 0.5 μg/ml puromycin.

To estimate the accumulated PDs at the time of clone isolation (considered to be the time at which the isolated clone reached confluence in a 100-cm dish), we assumed that the cell colonies that were picked were composed of ∼103 cells (10 PDs). Then the subsequent number of accumulated PDs until confluence was reached in a 100-cm dish was calculated using the formula ΔPDL = log (nf − n0)/log2; where n0 is the initial number of cells and nf is the final number of cells. Most clones reached confluence at 16–24 PDs.

Proliferation Assay

Cell proliferation was analyzed using the Cell Proliferation ELISA/ bromodeoxyuridine (Roche Diagnostics, Mannheim, Germany) and a [3H] incorporation assay. Cells (3 × 103) were plated in round-bottomed, 96-well plates. After 24-h [3H]thymidine pulse, cells were harvested onto glass fiber strips using an LKB Wallac 1295-001 Cell Harvester. [3H]thymidine incorporation of each cell lysate was determined in a Rackbeta Liquid Scintillation Counter (ICN, Costa Mesa, CA). Each sample was processed in triplicate.

Telomerase Activity Assay

Telomerase activity was measured using a modified version of the TRAP assay (5) . In some cases, an IC for PCR efficiency was included in the TRAP assay (TRAPeze kit, Oncor). Alternatively, the Telomerase PCR ELISA Kit (Roche Diagnostics) was used to detect PCR products, according to the manufacturer’s instructions.

Telomere Length Analysis

Southern Blot TRF Analysis.

Genomic DNA was extracted with a DNA Extraction Kit (Qiagen, Hilden, Germany). Genomic DNA (10 μg) was digested overnight with 10 units each of HinfI and RsaI (MBI Fermentas, Vilnius, Lithuania), loaded onto a 0.7% agarose gel, and transferred to a nylon membrane (Amersham). DNA fragments were hybridized to a digoxigenin-labeled telomere probe (TTAGGG)4. Telomeric sequences were detected by chloro-substituted 4t-dioxetane, chemiluminiscent substrate for alkaline phosphatase-star chemiluminescence (Roche Diagnostics).

Flow-FISH.

Telomere length was also measured by Flow-FISH as described (40) , for which 106 cells were used in each case. To normalize Flow-FISH data, two mouse leukemia cell lines (LY-R and LY-S) of known telomere lengths (9 and 40 kb, respectively; Ref. 41 ), were used as ICs. The telomere fluorescence was measured of at least 2 × 103 cells gated at G1-G0 cell cycle stage using a Coulter Flow EPICS XL cytometer with the SYSTEM 2 software (Coulter, Miami, FL). The mean telomeric fluorescence was calculated after subtracting the background from the probe alone.

Genetic Instability

Between 80–100 metaphases of each clone were scored for telomere fusions, chromatid breaks, and chromosome fragments by superimposing the telomere image on the 4′6-diamidino-2-phenylindole chromosome image in the TFL-telo program. Metaphase chromosomes were prepared by treatment of the cells with 0.1 μg/ml Colcemid for 4 h, followed by hypotonic lysis in 0.06 mmol KCl and fixation with methanol/acetic acid. After preparing metaphase slides, FISH was performed as described (10) .

In Vivo Tumor Formation Experiments

Cells (105) were injected s.c. in two leg sites of BALB/c nu/nu immunodeficient mice (Harlan, Barcelona, Spain). Tumor size was determined every second day from day 12 postinoculation.

Quantitative Detection of Wt mTERT and Mt mTERT-DN Transcript Levels Using the Light-Cycler PCR

Total RNA was extracted by RNAzol (WAK Chemie, Germany) and the reverse transcriptase reaction performed using 200 ng of RNA in a one-step RT-PCR. The RT-PCR reactions were done using the Light Cycler Instrument (Roche) and the LightCycler-RNA Amplification Kit SYBR Green I (Roche). The RT-PCR reactions were carried out using sets of primers specific for either the endogenous Wt (Wt primers) or the Mt mTERT-DN sequence (Mt primers; specific for the Mt sequence). The primers used to detect the endogenous Wt TERT were: wt-mTERTup (5′-TTT TGT TGA TGA CTT TCT-3′) and mTERTlow (5′-TCT GGG CAT AAC CTG AGT-3′). The primers to detect the Mt TERT were: mt-mTERTup (5′-TTT TGT TGC TGC CTT TCT-3′) and mTERTlow (5′-TCT GGG CAT AAC CTG AGT-3′). As an IC, β-actin-specific primers UBA-U144 (5′-GTG GGG CGC CCC AGG CAC CA-3′) and LBA-660 (5′-CTC CTT AAT GTC ACG CAC GAT TTC-3′) were used to calculate the relative concentration. For measurement of relative concentrations, a melting curve analysis of the amplified products was performed. The melting curve allows identification of the specific products as well as peak area calculations. To calculate the relative abundance of specific mTERT products, the peak area of the mTERT products was corrected by that of IC products.

RESULTS AND DISCUSSION

mTERT Aspartic Acid Mt Acts as a Dominant Negative of Mouse Telomerase.

In budding yeast and in man, telomerase activity can be abolished by replacement of any of the three conserved aspartic acid residues of amino acid motifs A and C of the TERT subunit (12 , 36 , 37) . Ectopic expression of these Mt hTERT in human cells results in telomerase inhibition, telomere shortening, and apoptosis (36 , 37) . These Mt hTERT subunits therefore act as dominant negatives of Wt telomerase, possibly by sequestering Wt hTerc into inactive telomerase complexes. To obtain dominant negative mutants of mouse TERT, the full-length mTERT cDNA and substituted amino acids Asp (861) and Asp (862) in conserved motif C to Ala residues (see “Materials and Methods”). This Mt murine TERT was termed mTERT-DN. mTERT-DN was cloned in a pBabe retroviral vector containing a puromycin selection marker (see “Materials and Methods”). The empty retroviral vector and that harboring the mTERT-DN were named pBabe and pDN, respectively.

To study the effect of mTERT-DN expression on telomerase activity, mouse RenCa kidney carcinoma tumor cells (39) were transduced with either the empty pBabe vector or the pDN vector (see “Materials and Methods”). Three days after retroviral infection, puromycin selection was started and maintained throughout the time of the experiment. Forty-one pBabe-transduced clones and 46 pDN-transduced clones were isolated. Whereas all pBabe-transduced clones (41 of 41) grew successfully in culture, only 66% (31 of 46) of pDN-transduced clones could be cultured after isolation, suggesting an early loss of viability in the pDN clones (see below). Accumulated PDs for all clones at the time of isolation were calculated to range from ∼16–24 PD, depending on the clone (see “Materials and Methods”). Telomerase activity levels were determined in all clones at the time of isolation, as well as at later passages (see below), using conventional TRAP assays and the Telomerase PCR ELISA Kit (see “Materials and Methods”). All of the pBabe-transduced clones showed telomerase activity levels similar to those of the parental RenCa cells (Fig. 1 ⇓ shows representative examples of TRAP activity for RenCa cells, as well as for pBabe and pDN-transduced clones). Telomerase activity diminished to undetectable levels in all clones tested at early PD, indicating that mTERT-DN acts as a dominant negative inhibitor of Wt mouse telomerase activity (see Fig. 1 ⇓ for representative examples).

Fig. 1.
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Fig. 1.

Inhibition of telomerase activity by expression of a dominant negative Mt of mTERT. S-100 extracts were prepared from pBabe-transduced (pBabe1, pBabe3, pBabe4, pBabe5, pBabe6, and pBabe7), pDN-transduced (pDN1, pDN2, pDN3, pDN5, pDN6, pDN7, pDN9, and pDN12), and control RenCa cells and assayed for telomerase activity using the TRAP assay (see “Material and Methods”). Extracts were pretreated (+) or not (−) with RNase. In the case of clones pDN2 and pDN12, RNase-treated extracts showed background levels of telomerase activity, which were never detected in the untreated lanes. Number of cells or protein concentration/reaction is indicated.

Telomere Length in Murine Cells Expressing a Dominant Negative TERT.

We measured telomere length in both pBabe and pDN-transduced RenCa clones at different PDs after clone isolation, using both conventional telomeric TRF by Southern blot (5) and telomeric FISH using flow cytometry (Flow-FISH; Ref. 40 ). All pDN clones analyzed (pDN7, pDN10, pDN12, pDN23, and pDN27) except pDN8 had smaller TRFs than a control pBabe clone (pBabe7; Fig. 2A ⇓ . Whereas the pBabe7 clone shows telomeres with a mean TRF length of 7 kb, TRFs from six independent pDN clones ranged from 2–4 kb in length at the time of clone isolation, suggesting that telomere shortening had occurred in the pDN clones relative to the pBabe clone. The pBabe7 clone showed a stable TRF length of 7 kb with increasing PDs, in agreement with the presence of telomerase activity (Fig. 2A) ⇓ . Flow-FISH analysis was used for quantitative measurement of telomere length. Flow-FISH allows estimation of the length of TTAGGG repeats as the fluorescence intensity of telomeres in interphase nuclei previously hybridized with a fluorescent PNA-telomeric probe (see “Materials and Methods”). Fig. 2B ⇓ shows the telomeric fluorescence of a pBabe-transduced clone (pBabe3) at different PDs, the telomere fluorescence of the parental RenCa cell line, as well as that of a control cell line (LY-S cells; see “Materials and Methods”) with a known telomere length of 7 kb. At later passages after clone isolation, two different pDN clones (pDN1 and pDN2) showed lower telomere fluorescence than the pBabe3 clone, in agreement with telomere shortening in these cells (Fig. 2B) ⇓ . Intriguingly, after the initial shortening, telomere length was maintained or slightly increased with increasing PDs in some of the pDN clones (pDN7, pDN10, and pDN23; Fig. 2A ⇓ ), whereas other clones showed further telomere shortening (pDN1 and pDN2; Fig. 2B ⇓ ; see next section for discussion). Fig. 2C ⇓ shows representative images of metaphases from pBabe3 and pDN1 clones at two different PDs (early and late PDs) previously hybridized with a fluorescent PNA-probe against telomeres. Cytogenetic analysis of metaphases from pBabe3 and pDN1 clones revealed that chromosomal instability with increasing PDs was similar in both clones (not shown).

Fig. 2.
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Fig. 2.

Telomere length measurements in pBabe- and pDN-transduced RenCa clones. A, TRF length determinations by Southern blot of pBabe and pDN-transduced clones. A pBabe-transduced clone (pBabe7) and several pDN-transduced clones (pDN7, pDN8, pDN10, pDN12, pDN23, and pDN27) at several different PDs, as well as a tumor-derived cell line (pDN7-T1) were used for TRF analysis. Note that TRF signals are lower (2–4 kb) in all pDN-transduced clones (except pDN8) than in the control pBabe-transduced clone (7 kb). B, telomere length expressed as arbitrary units of fluorescence as determined by Flow-FISH (see “Materials and Methods”) in a pBabe-transduced (pBabe3) and two pDN-transduced (pDN1 and pDN2) clones. C, representative images of metaphases from early and late PD pBabe-transduced (pBabe3), pDN-transduced (pDN1) and control RenCa cells that have been hybridized with a fluorescent PNA-telomeric probe (light dots; see “Materials and Methods”); chromosomes are stained with DAPI (gray area).

All together, these data indicate that telomerase inhibition via expression of mTERT-DN results in initial telomere shortening in the pDN-transduced clones, as previously described for human cells (36 , 37) ; however, some pDN clones showed telomere maintenance or elongation at later PD after clone isolation (see below).

Reactivation of Telomerase Activity in pDN Clones with Increasing PDs.

The fact that some pDN clones did not show continued telomere shortening with increasing PDs (Fig. 2A) ⇓ prompted us to monitor telomerase activity with increasing PDs in all pDN clones. Telomerase activity was analyzed using both the conventional TRAP assay (see Fig. 3A ⇓ for representative examples) and the telomerase PCR ELISA Kit (see Fig. 3B ⇓ for representative examples). A control pBabe4 clone showed similar telomerase activity levels throughout the different PDs analyzed, as determined both by TRAP and the PCR ELISA Kit (Fig. 3, A and B) ⇓ . Strikingly, all pDN clones analyzed reactivated telomerase activity to different levels with increasing PDs after clone isolation (Figs. 3A and 3B ⇓ show representative examples). Clone pDN2, in particular, showed very low telomerase activity at isolation (PD24), although activity had already recovered to normal levels at PD32 and was maintained thereafter (Fig. 3A ⇓ ; we cannot rule out that telomerase had reactivated before PD32 in this clone, because PD32 was the first PD analyzed after PD24). pDN1 and pDN 5 clones also showed up-regulation of telomerase activity with increasing PDs (Fig. 3B) ⇓ . Curiously, telomerase up-regulation in pDN clones was not always sufficient to prevent telomere shortening with increasing PD (i.e., clones pDN1 and pDN2; Fig. 2B ⇓ ). This can be explained by insufficient telomerase reactivation to prevent telomere shortening in these particular clones.

Fig. 3.
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Fig. 3.

Reactivation of telomerase activity in pDN-transduced clones with increasing PDs. A, S-100 extracts were prepared at different PDs of a pBabe-transduced (pBabe4) and a pDN-transduced (pDN2) clone and assayed for telomerase activity using the TRAP assay (see “Material and Methods”). Extracts were pretreated (+) or not (−) with RNase. Protein concentration/reaction are indicated. An IC for PCR efficiency is included (IC). B, S-100 extracts were prepared from different PDs pBabe-transduced (pBabe4; ▪), pDN-transduced (pDN1, pDN5; □), and several derived tumor cell lines (pBabe4T1; ▪; pDN1T1, pDN1T2, and pDN5T1; □). For telomerase detection, the Telomerase ELISA PCR Kit was used (see “Materials and Methods”). Levels of telomerase activity are expressed relative to that of RenCa ( Embedded Image) cells, which is considered 1 arbitrary unit.

Quantification of Wt mTERT and mTERT-DN Transcripts with Increasing PDs in pBabe and pDN-transduced Clones.

The efficient reactivation of telomerase activity in the pDN-transduced clones cultured under puromycin selection suggests that it was independent of the presence of the pDN construct. We thus developed an RT-PCR-based method to quantify endogenous mTERT and mTERT-DN mRNA levels. Total RNA from the different clones was used for a one-step RT-PCR using the Light Cycler (see “Materials and Methods”), using sets of primers that were specific for either the mTERT-DN (Mt primers) or the endogenous TERT (Wt primers; see “Materials and Methods”). As shown in Fig. 4A ⇓ , at 65°C and using the Mt primers, the mTERT-DN transcripts were detected in a pDN clone (pDN2 at PD24 and PD47) and in a pDN2-derived tumor cell line (pDN2 T) but not in a pBabe clone (pBabe3 at PD45), in a pBabe3-derived tumor cell line (pBabe3 T), or in the RenCa cells, indicating that the Mt primers are specific for the mTERT-DN sequence and do not recognize the endogenous mTERT (see Fig. 4A ⇓ ). The Wt primers at 65°C did not detect any products (see below for RT-PCR at lower temperatures). For measurement of relative concentrations, a melting curve analysis of the amplified products was performed. The melting curve allows identification of the specific products as well as peak area calculations. Fig. 4A ⇓ shows the quantification of the Mt mTERT-DN levels as peak area units, which have been previously corrected by the IC signal (see “Materials and Methods”). Fig. 4A ⇓ also shows an agarose gel with the mTERT-DN specific bands only present in the pDN-transduced clones. Fig. 4B ⇓ shows mTERT-DN mRNA levels relative to those of the IC in several pDN-transduced clones at two different PDs (pDN1, pDN5, pDN6, pDN7, pDN8, pDN9, pDN10, pDN12, pDN23, and pDN27). Quantitation of the mTERT-DN specific product in all these different pDN clones indicated that the amounts did not significantly change when comparing early with late PD, suggesting that expression of the Mt TERT mRNA is maintained in the pDN-transduced cells (Fig. 4B) ⇓ .

Fig. 4.
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Fig. 4.

Detection and quantitation of endogenous mTERT and Mt mTERT-DN transcript levels by Light-cycler PCR. A, to quantify endogenous mTERT and Mt mTERT-DN levels in pDN clones at early and late PD, we used Light-Cycler PCR (see “Materials and Methods”). Mt primers: specific for mTERT-DN sequence; Wt primers: specific for the mTERT sequence. At 65°C and using the Mt primers, the mTERT-DN transcripts are detected in a pDN clone (pDN2 at PD24 and PD47), and in a pDN-derived tumor cell line (pDN2 T) but not in a pBabe clone (pBabe3 at PD45), in a pBabe3-derived tumor cell line (pBabe 3 T) or in the RenCa cells. An agarose gel with the Light-Cycler RT-PCR products is also shown. Melting curves used for quantification of the mTERT-DN specific products are also shown. Quantification of the mTERT-DN specific product is shown as peak area units previously corrected by the IC values. The mTERT-DN mRNA amounts did not change when comparing early and late PDs. B, quantification of mTERT-DN mRNAs levels at different PDs in several pDN-transduced (pDN1, pDN5, pDN6, pDN7, pDN8, pDN9, pDN10, pDN12, pDN23, and pDN27) clones. Mt primers were used for all reactions at an annealing temperature of 65°C. Quantification of the mTERT-DN specific product in the pDN clone indicated that the amounts did not change when comparing early and late PDs. mTERT-DN levels are expressed relative to those of the IC. C, quantification of endogenous mTERT mRNA levels at different PDs in pDN-transduced clones (pDN2 and pDN5 as well as the corresponding tumor cell lines pDN2-T and pDN5-T, respectively), as well as in pBabe-transduced clones (pBabe3 and the pBabe3-T tumor cell line and clone pBabe4) and in the parental RenCa cell line. Light Cycler reactions were performed at three different temperatures (52, 58, and 65°C). The RT-PCR products run in an agarose gel are also shown. Arrow, Wt mTERT-specific band. Wt primers were used for all reactions.

The endogenous mTERT mRNA was detected using Wt primers in all pBabe and pDN-transduced clones tested, as well as in the parental RenCa cell line (Fig. 4C ⇓ ; see results at 52°C and 58°C). Strikingly, the endogenous mTERT transcript levels were always higher (∼2-fold) in the pDN-transduced clones than in the pBabe-transduced clones or in the RenCa cells at all different temperatures tested (see Fig. 4C ⇓ for representative examples; this difference was statistically significant as determined by Student’s t test; P < 0.01), suggesting that endogenous mTERT expression is up-regulated in the pDN clones as compared with the pBabe clones. The endogenous mTERT mRNA up-regulation in the pDN clones preceded telomerase reactivation (i.e., clone pDN2 showed increased mTERT mRNA levels at PD24 when telomerase activity was still low; Fig. 4C ⇓ ).

All together, these results indicate that loss expression of the pDN constructs is not the cause of the elevation in telomerase activity with increasing PDs in the pDN-transduced clones. The efficient reactivation of telomerase activity in all pDN clones coincides, however, with a significant up-regulation of the endogenous mTERT mRNA levels, suggesting that this is one of the possible mechanisms by which telomerase activity is reactivated in these clones. These results have important implications regarding the use of murine cells to screen for telomerase inhibitors.

Effect of mTERT-DN Expression on Cell Viability.

During the process of clone isolation or shortly thereafter, some of the pDN clones contained cells with abnormal morphology (flat and enlarged cells showing a senescence-like phenotype). Fig. 5A ⇓ shows representative images of these senescent-like cells in clones pDN2 and pDN7, as well as a control pBabe4 clone with normal cell morphology. In addition, 32% of the pDN clones did not survive culture after clone isolation, suggesting the loss of viability in these clones. To study this, we performed [3H]thymidine incorporation assays in pBabe and pDN-transduced clones at the time of isolation. pDN clones showed, on average, a 30% decrease in proliferative capacity compared with simultaneously isolated pBabe clones (Fig. 5B) ⇓ . It is interesting to note that those pDN clones that survived the isolation process showed no loss of viability at later PDs. This is in marked contrast to previous descriptions of human clones expressing dominant negative hTERT, which showed a dramatic loss of viability (36 , 37) .

Fig. 5.
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Fig. 5.

Cell viability of pDN-transduced RenCa clones. A, representative images from a pBabe (pBabe4 PD36)- and two pDN-transduced (pDN2 PD35 and pDN PD37) clones. Notice the senescent-like morphology of the pDN-transduced clones. B, average [3H]thymidine incorporation in pDN and pBabe clones at the time of clone isolation. Bars, SD.

Tumor Formation in Vivo by mTERTDN-transduced RenCa Cells.

To study the effect of murine telomerase inhibition on tumor formation in vivo, early PD pBabe (pBabe1, pBabe4, and pBabe7) and pDN-transduced clones (pDN2, 3, 6, and 7) were injected s.c. into immunodeficient mice, and tumor formation was monitored over a 32-day period (Fig. 6 ⇓ ; see “Materials and Methods”). No differences in tumor latency or tumor size were observed between pBabe- and pDN-injected mice. The lack of inhibitory effect of the dominant negative mTERT mutation on tumor growth in mice is likely to be the consequence of telomerase reactivation in the mTERTDN-transduced clones (see above). Telomere length and telomerase activity were measured in several cell lines derived from pDN-induced tumors: pDN1T1 and pDN1T2 were derived from two independent tumors produced by the pDN1 clone; pDN5T1 was derived from a tumor produced by the pDN5 clone; pDN7T1 was derived from a tumor produced by the pDN7 clone; and pBabe4T1 was derived from a tumor produced by the pBabe4 clone. Telomerase activity was detected in all pBabe and pDN-derived tumor lines (see Fig. 3B ⇓ ), in agreement with reactivation of telomerase activity in the injected pDN cells in vivo. On the other hand, the pDN7-T1 tumor cell line showed a TRF length similar to that of the parental pDN7 clone, also suggesting that telomeres have been maintained in this tumor in vivo (Fig. 2A ⇓ ) and confirming the presence of telomerase activity in the pDN-induced tumors despite the expression of the mTERT-DN construct as shown in Fig. 4 ⇓ .

Fig. 6.
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Fig. 6.

In vivo tumor formation by pDN-transduced RenCa clones. Tumor growth during increasing days after the injection of early PD pBabe-transduced (pBabe1 PD36, pBabe4 PD24, and pBabe7 PD24) and pDN-transduced (pDN2 PD24, pDN3 PD24, pDN6 PD 30, and pDN7 PD30) cells into nude mice.

Conclusions.

Here we show that ectopic expression of a dominant negative Mt of murine TERT in the RenCa renal carcinoma cell line, which has telomeres similar in length to those of human cells, results in initial telomerase inactivation and telomere shortening. In contrast to inhibition of human telomerase using hTERT dominant negative mutants, however, telomerase activity was efficiently reactivated in the RenCa clones expressing the dominant negative mTERT. Furthermore, we observed no loss of viability or impact on tumor formation by these cells in long-term assays. All together, these results indicate that telomerase inactivation in RenCa tumor cells by mTERT dominant negative expression does not prevent tumor formation attributable to a rapid reactivation of telomerase activity, even in the presence of the Mt mTERT-DN mRNA. Using quantitative Light-Cycler RT-PCR, we have also established that this telomerase activity reactivation is mediated by an up-regulation of the endogenous mTERT mRNA levels. It is likely that the pressure to maintain telomeres in these cells may provoke telomerase up-regulation, which overcomes the presence of the Mt mTERT-DN transcript. A plausible mechanism for this efficient telomerase up-regulation may be amplification of the endogenous mTERT gene, as recently described for the hTERT gene in human tumors (42) . In summary, the results presented here contrast with those published for telomerase inhibition in human tumor cell lines and suggest the existence of important differences in telomerase regulation between human and murine cells.

Acknowledgments

We thank Catherine Mark for proofreading the manuscript.

Footnotes

  • The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 Work at the Freie Universität is supported by Grant HE 1362/6-1,2 of the Deutsche Forschungsgemeinschaft. E. S-G. and E. S. are supported by the Spanish Ministry of Health (Fondo de Investigaciones Sanitanas) and by the Government of Madrid (Comunidad Autonoma de Madrid), respectively. M. A. B.’s laboratory of is funded by The Swiss Bridge Award 2000 to M. A. B., Spanish Ministry of Science and Technology PM97-0133, the CAM 08.1/0030/98, the European Union (FIGH-CT-1999-00009, FIGH-CT-1999-00002, QLG1-1999-01341), and by the Department of Immunology and Oncology (DIO). The Department of Immunology and Oncology was founded and is supported by the Spanish National Research Council (CSIC) and by the Pharmacia Corporation.

  • ↵2 To whom requests for reprints should be addressed, at Department of Immunology and Oncology, Lab 413, National Centre of Biotechnology, CSIC, Campus de Cantoblanco, Madrid E-28049, Spain. Phone: 34-91-5854846; Fax: 34-91-372 0493; E-mail: mblasco{at}cnb.uam.es

  • 3 The abbreviations used are: TERT, telomerase reverse transcriptase; Terc, telomerase RNA component; PD, population doubling; IC, internal control; TRAP, telomeric repeat amplification protocol; TRF, telomeric restriction fragment; FISH, fluorescence in situ hybridization; RT-PCR, reverse transcription-PCR; Wt, wild-type; Mt, mutant; pDN, pBabe containing mTERT-DN; pBabe, empty pBabe plasmid.

  • ↵4 E. González-Suárez and M. A. Blasco, unpublished results.

  • Received November 6, 2000.
  • Accepted May 8, 2001.
  • ©2001 American Association for Cancer Research.

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Cancer Research: 61 (14)
July 2001
Volume 61, Issue 14
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Telomerase Inhibition in RenCa, a Murine Tumor Cell Line with Short Telomeres, by Overexpression of a Dominant Negative mTERT Mutant, Reveals Fundamental Differences in Telomerase Regulation between Human and Murine Cells
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Telomerase Inhibition in RenCa, a Murine Tumor Cell Line with Short Telomeres, by Overexpression of a Dominant Negative mTERT Mutant, Reveals Fundamental Differences in Telomerase Regulation between Human and Murine Cells
Jana Sachsinger, Eva González-Suárez, Enrique Samper, Rüdiger Heicappell, Markus Müller and María. A. Blasco
Cancer Res July 15 2001 (61) (14) 5580-5586;

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Telomerase Inhibition in RenCa, a Murine Tumor Cell Line with Short Telomeres, by Overexpression of a Dominant Negative mTERT Mutant, Reveals Fundamental Differences in Telomerase Regulation between Human and Murine Cells
Jana Sachsinger, Eva González-Suárez, Enrique Samper, Rüdiger Heicappell, Markus Müller and María. A. Blasco
Cancer Res July 15 2001 (61) (14) 5580-5586;
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