Telomerase enzymatic component hTERT shortens long telomeres in human cells (2024)

hTERT decreases the length of long telomeres and increases the length of short telomeres in ALT+ cancer cell lines

ALT+ cancer cells are characterized by having excessively long and heterogeneous telomere lengths across all chromosomal ends without telomerase activity^6-8. To examine the effects of hTERT in cells with excessively long telomeres, a haemagglutinin (HA)-tagged hTERT was ectopically expressed in U2OS, an osteosarcoma cell line with high ALT activity (Table 1; Fig. S1). Telomere length (TL) at each of the chromosomal ends was measured by telomere fluorescent in situ hybridization (T-FISH) as previously described¹⁴ and compared between hTERT-expressing cells and vector control cells. Dramatic decrease in the average telomere length (Avg_TL) per telomere was observed in U2OS–hTERT cells compared with that in U2OS–vector control cells (6237 vs. 2314, P < 0.001; Table 1; Fig.1A and B; Fig. S2A). Significant decreases in the telomere length variation (TLV), defined as co-efficient of variation (CV%) of all measured telomeres, were observed (76.9 vs. 61.1, P < 0.001; Table 1). The frequency of excessively long telomeres, defined as TL > 3× average TL for a given cell type, were also seen (average 34 per cell in vector control cells vs. 0.4 per cell in hTERT-expressing cells, P < 0.001). Correlation analysis revealed a strong inverse correlation between the lengths of TL at each chromosomal end in vector control cells and the percentage TL change at the corresponding chromosomal end of U2OS–hTERT cells (r = −0.91, P < 0.001; Fig. S3A). The most striking observation is the significant TL shortening at most of chromosomal ends in U2OS-hTERT cells compared with those in U2OS–vector control cells (Fig.1A and B). No significant increases in TL were observed for any of the chromosomal ends. In addition, telomere restricted fragment (TRF) analysis verified these results, as there was a significant decrease in telomere length upon hTERT expression in U2OS cells (Fig. S3B). These data suggested that the dominant effect of hTERT expression in U2OS cells was shortening the excessively long telomeres.

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Figure1. Expression of hTERT shortens long telomeres and elongates short telomeres in ALT+ cancer cells. Empty vector, WT or mutant hTERT, or hTR was expressed in ALT+ U2OS cells (A–D), SAOS2 cells (E–H), or a hTR-negative cell line WI38-VA13 (I and J). Metaphase spreads were prepared and hybridized with a Cy3 (red) telomere PNA probe and FITC (green) label chromosome-specific centromere probes. Thirty cells were analyzed per cell line, and representative metaphase images were displayed. (A) U2OS-vector, (B) U2OS-hTERT, (C) U2OS-mutant-hTERT, (D) U2OS-hTR; Amplified chromosome X was showed in the upper-right corner. (E) SAOS2-vector, (F) SAOS2-hTERT, (G) SAOS2-mutant-hTERT, (H) SAOS2-hTR; amplified chromosome 11 is shown in the upper right corner. (I) WI-38-VA13 vector, and (J) WI-38-VA13-hTERT.

We further examined specific chromosome ends possessing long or short TL. In U2OS cells, there is one normal X chromosome. Its long arm (Xq) has the shortest telomere length, while its short arm (Xp) has an excessively long telomere (Fig.1B). We found that expression of hTERT significantly decreased the average TL on Xp (9825 vs. 3141, P < 0.001), while it had no significant effect on the TL of Xq (1480 vs. 1747, P = 0.49; Table 1; Fig.1A and B), indicating that hTERT selectively shortens long telomeres in U2OS cells.

hTERT-mediated shortening of excessively long telomeres was confirmed in another ALT+ cancer cell line, SAOS2. This cell line has low ALT activity and is characterized by having short TL at the most chromosomal ends and one excessively long telomere on the long arm (q) of one of chromosomes 11 (Fig.1E). Expressing hTERT in SAOS2 induced a significant decrease in TLV and increase in the average TL per telomere (Table 1; Fig. S2B). Correlation analysis revealed a strong inverse correlation between the lengths of TL at each chromosomal end in vector control cells and the percentage TL change at the corresponding chromosomal end of SAOS2–hTERT cells (r = -0.84, P < 0.001; Fig. S3C). Most importantly, we found that expression of hTERT significantly shortened TL on 11q (−56%) and elongated TL on 11p (+248%; Table 1; Fig.1E and F), confirming that hTERT selectively shortens long telomeres and elongates short telomeres in ALT+ cancer cells. Moreover, indirect immunofluorescence and telomere FISH displayed the localization of hTERT at those telomeres with high signal intensity, suggesting that hTERT can be recruited to long telomeres (Fig. S4).

Together, these data indicate that hTERT plays dual roles in telomere length regulation by shortening excessively long telomeres and elongating short telomeres, thus maintaining optimal telomere length at each of the chromosomal ends for efficient protection. The data also indicate that the switch between the 2 opposite hTERT functions is dictated by the length of the telomere at a specific chromosomal end.

Expression of hTERT in human primary fibroblasts shortens long telomeres and elongates short telomeres

Wild-type hTERT was overexpressed in IMR90 (primary human fibroblasts) at population doubling (PD) 10, and increased telomerase activity and hTERT expression were confirmed by the qRT-PCR, western blot analysis and the TRAP assay (Table 1; Fig. S1). Cells were harvested at passage 3 after hTERT transfection, following a standard cytogenetic protocol for chromosome preparation. The average TL per telomere was significantly increased in hTERT-expressing cells compared with that in vector control cells (Table 1; Fig.2A–C). We also noticed a significant decrease in TLV across all chromosomal ends (Table 1) and a significant decrease in the frequency of excessively long telomeres (average 2.6 per cell in vector controls vs. 1.6 per cell in hTERT-expressing cells, P = 0.005). This observation prompted us to examine if changes in TL at each chromosomal end in hTERT-expressing cells were correlated with the TL of the corresponding chromosomal end in vector control cells. Surprisingly, we found a strong inverse correlation between the TL in vector control cells and the percentage TL change in hTERT-expressing cells (Pearson correlation coefficient [r] = −0.80, P < 0.001; Fig.2A). More importantly, the data indicated that telomere length at the chromosomal end possessing the longest telomere (10 q) was significantly shortened in hTERT-expressing cells compared with that in control cells (−18%, P = 0.03; Table 1), while telomere length at the chromosomal end possessing the shortest telomere (19p) showed significant elongation in hTERT-expressing cells compared with that in controls (+70%, P < 0.001; Table 1). In IMR90 cells, 6 out of 46 (13%) chromosomal ends showed shortened average TL in IMR90-hTERT cells (Fig.2A).

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Figure2. Expression of hTERT shortens long telomeres and elongates short telomeres in human primary fibroblasts. Expression of hTERT in human primary fibroblasts IMR90 and WI38 shortened long telomeres and elongated short telomeres. Metaphase spreads were prepared and hybridized with a Cy3 (red) telomere PNA probe and FITC (green) label chromosome specific centromere probes. Figure1A and D show correlations between the average TL at specific chromosomal ends in vector control cells and percents of TL change at the corresponding chromosomal ends in hTERT-overexpressing cells. Each dot represents a chromosomal end. Thirty cells were analyzed per cell line. Representative metaphase images were displayed. (B) IMR90-vector, (C) IMR90-hTERT, (E) WI38-vector, (F) WI38-hTERT.

Similar results were obtained with another primary human fibroblast cells (WI38), showing significant decrease in TLV across all chromosomal ends (Table 1; Fig.2D–F) in hTERT-expressing cells compared with control cells and strong inverse correlation between the lengths of TL at each chromosomal end in vector control cells and percentage TL change at the corresponding chromosomal end of hTERT-expressing cells (r = −0.66, P < 0.001; Fig.2D). A significant decrease (−43%) in average TL at Xp (longest telomere in WI38) and an increase (+51%) in average TL at 21q (the shortest telomere in WI38) were also observed (Table 1) upon expressing hTERT. Fifteen out of 46 (33%) chromosomal ends showed shortened average telomere lengths in WI38-hTERT cells (Fig.2D). These data suggested that hTERT plays a dual role in the regulation of telomere length by shortening the long telomeres and extending the short telomeres in normal human fibroblasts.

Upregulation of endogenous hTERT maintains small TLV in primary epithelia cells

Fibroblast feeder cells, in combination with a Rho kinase inhibitor, have been shown to immortalize human primary epithelia cells that retained a normal karyotype and remained non-tumorigenic.¹⁵ This culture condition dramatically upregulates endogenous hTERT expression level (8- to 14-fold increase) and telomerase activity.¹⁵ Under the feeder layer co-culture condition, GUMC001 cells (human primary prostate epithelia cells) maintained relatively small and constant TLV and no significant change in average telomere length per telomere from PD6 to PD48, whereas the number of excessively long telomeres remained stably low, and the number of very short telomeres decreased from PD6 to PD48 (Table 2). The observed small TLV and low number of excessively long and very short telomeres in GUMC001 cells are comparable with what was observed in hTERT-expressing IMR90 cells (Table 1). In contrast, IMR90 (human primary fibroblast cells) cultured in conventional condition resulted in rapid loss of telomere length and significantly increased TLV from PD10 to PD60. The number of excessively long telomeres also showed a significant trend of increase (P for trend < 0.001, Table 2). IMR90 cells grown in conventional cell culture condition typically have very low levels of hTERT expression and telomerase activity (Table 2). These data suggest that upregulation of endogenous hTERT in prostate epithelial cells may have shortened excessively long telomeres and elongated short telomeres to maintain a small TLV, which is consistent with the results obtained by ectopically overexpressing hTERT in human primary fibroblast cells. Because GUMC001 does not grow in conventional cell culture conditions without feeder layer, we could not obtain chromosome preparations without hTERT upregulation to show a direct comparison.

hTERT knockdown in telomerase-positive cancer cells decreases the telomere length

To investigate the effects of downregulation of hTERT on telomere length in telomerase-positive cancer cells, we used siRNA to knockdown hTERT in MDA231, a breast cancer cell line. hTERT knockdown efficiency was confirmed by TRAP assay and qRT-PCR. Downregulation of hTERT significantly decreased average telomere length per telomere and increased TLV in MDA231 cells. The increase in TLV is accompanied by increases in the frequency of very short telomeres and excessively long telomeres (Table 2). Similar results were obtained for another telomerase-positive fibrosarcoma cell line, HT1080 (Table 2). Our data indicates that knockdown hTERT in telomerase-positive cancer cell lines affects telomere elongation and results in shorter telomeres across all chromosomal ends.

The enzymatic domain of hTERT is required for hTERT-mediated telomere shortening

To investigate the role of telomerase activity in hTERT-mediated telomere shortening, we used a haemagglutinin (HA)-tagged hTERT mutant construct in which the aspartic acid and valine residues at positions 710 and 711 in the third reverse transcriptase motif of hTERT were substituted with alanine and isoleucine, respectively.¹⁶ This mutant construct lacks telomerase activity (enzymatically dead) and has a dominate-negative effect on telomere elongation. Consistent with previous reports,^16,17 no telomerase activity was detected in HA immunoprecipitates after the mutant construct was expressed (Fig. S1). Expressing this mutant hTERT in U2OS cells did not affect average TL per telomere and TLV (Table 3; Fig.1C). We observed no significant change in TL on both Xp (long telomere) and Xq (short telomere; Table 3; Fig.1C). These data suggest that the catalytic domain of hTERT is required for shortening the excessively long telomeres.

Expressing the mutant hTERT in SAOS2 cells induced a significant decrease in average TL per telomere and increase in TLV. The increase in TLV in cells expressing the mutant hTERT was primarily driven by significant increases in the frequency of very short telomeres (average 10.6 per cell in vector control cells vs. 16.3 per cell in hTERT-mutant expressing cells, P < 0.001) (Table 3). While short telomeres (i.e., 11p) had significantly decreased TL (−52%, P = 0.02), in contrast, excessively long telomere on 11q had slightly increased TL (+29%, Table 3; Fig.1G), confirming that this mutant hTERT failed to shorten excessively long telomeres. Together, our data indicated that the enzymatic domain is required for hTERT-mediated telomere shortening.

hTR is not required for hTERT-mediated telomere shortening

Previous reports showed that overexpression of hTR increased telomerase activity and elongated telomere length.¹¹ To examine the effect of hTR on telomere shortening, we expressed hTR in U2OS and SAOS2 cells. The function of hTR construct was first tested by expressing it in HT1080, and hTR expression resulted in 8- and 6-fold increase in hTR mRNA level and telomerase activity, respectively, consistent with previous reports.^11,12

Expressing hTR in U2OS cells significantly increased average TL per telomere and borderline significantly decreased TLV, due to a decrease in the frequency of very short telomeres. Although hTR expression significantly increased telomere length on short telomeres (Xq, +436%), it had no significant effect on the length of the long telomeres on Xp (Table 3; Fig.1D). Expressing hTR in SAOS2 cells did not shorten the telomere length on chromosome 11q (Table 3; Fig.1H), although hTR mRNA levels increased 6–8-fold (Fig. S1). These data indicate that overexpression of hTR alone does not shorten long telomeric DNA.

To further confirm whether hTR is required for hTERT-mediated telomere shortening, we expressed wide-type hTERT in a hTR-negative cancer cell line, WI38 VA13. T-FISH analysis revealed that expression hTERT dramatically shortened average telomere length per telomere and decreased frequency of excessively long telomeres from an average of 20 per cell in vector controls cells to 6 per cell in hTERT expression cells (P < 0.001, Table 3, Fig.1I and J), indicating that hTR is not required in hTERT-mediated telomere shortening.

TPP1 is required for hTERT-mediated telomere shortening

TPP1 is a shelterin protein and interacts with telomerase for telomere elongation.^18-23 We examined its role on hTERT-mediated telomere shortening. TPP1 knockdown did not affect average TL in SAOS2 (P = 0.72) and had no significant effect on the length of very long telomeres on 11q (Fig.3A–C). In TPP1-depleted SAOS2 cells, overexpressing hTERT significantly increased average TL per telomere (P < 0.001) and had no significant effect on TLV (P = 0.14). Analysis of chromosome-specific TL revealed that expression of hTERT did not shorten long telomeres on 11q in TPP1-depleted SAOS2 cells (Fig.3B and C), suggesting that hTERT-mediated telomere shortening requires TPP1.

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Figure3. TPP1 physically and functionally interacts with hTERT to regulate telomere length. (A) Depletion of TPP1 by shRNA. Lentiviral empty vector or TPP1 shRNAs were expressed in SAOS2 cells. Protein expression was analyzed by western blotting with indicated antibodies. (B) Effects of depletion of TPP1 on telomere length. hTERT was expressed in ALT+ SAOS2 cells with or without expression of TPP1 shRNA. Thirty cells were analyzed per cell line. (C) Representative metaphase images of SAOS2 cell with or without shTPP1 and/or hTERT expression. Amplified chromosome 11 was shown in the upper right corner. (D) TPP1 binds to hTERT in SAOS2 cells by coimmunoprecipitation. Nuclear extracts (0.5 mg) were treated with DNase I and then subjected to immunoprecipitation with the anti-Flag (TPP1) antibody, followed by western blotting with indicated antibodies. IgG was used as a control. Fifty micrograms of nuclear extract was loaded as input. (E) Recombinant TPP1 recruits hTERT binding to the telomere DNA substrate. The biotin-D-loop DNA was incubated with in vitro-translated HA-hTERT in the absence or presence of various amounts of recombinant TPP1 (5 and 20 nmol). The biotinylated DNA substrate and its bound proteins on the beads were analyzed by SDS-PAGE gel. Quantitative data is from 3 independent experiments (mean ± S.D.). Bovine serum albumin did not affect TPP1-enhanced hTERT binding to the D-loop substrate. HA-hTERT was detected by WB with the anti-HA antibody. Input, 50% of the total HA-hTERT used in each reaction. (F) hTERT generates t-circle formation, and TPP1 is required for hTERT-mediated t-circles. Empty vector and hTERT were expressed in ALT+ SAOS2 cells with or without expression of shTPP1, and 2D assay was performed to detect t-circle formation.

Using immunoprecipitation assays, we found that Flag-TPP1 co-precipitated with HA-hTERT (Fig.3D). Treatment with DNase-I did not prevent the TPP1–hTERT interaction, excluding bridging effects of nucleic acids. We then tested whether recombinant TPP1 regulates the binding of HA-hTERT to telomere DNA. Immunoblot analysis with anti-HA antibody showed that, in the absence of TPP1, HA-hTERT was associated with biotinylated-telomeric DNA substrates that were retrieved by streptavidin beads (Fig.3E). The addition of TPP1 significantly increased the binding of HA-hTERT to the telomeric DNA substrates in a dose-dependent manner (Fig.3E). Bovine serum albumin did not affect the telomere DNA binding activity of HA-hTERT. These data indicate that TPP1 dramatically enhances hTERT binding to the telomeric DNA substrates.

hTERT-mediated telomere shortening increases t-circle formation

T-circle formation has been shown to involve the telomere recombination mechanism,^13,24-26 and the increase in t-circles in ALT+ cells is considered a by-product of removed long telomeres.¹¹ Expression of hTERT in SAOS2 cells increased t-circles as measured by the 2D gel assay (Fig.3F). The increase in hTERT-induced t-circles was also confirmed in primary human fibroblasts (IMR90) by overexpression of hTERT (data not shown). Further, we found that TPP1 depletion prevented hTERT-induced t-circle formation in SOAS2 cells (Fig.3F), consistent with our T-FISH findings that hTERT overexpression failed to shorten long telomeres on 11q in TPP1-depleted SAOS2 cells (Fig.3B and C). Together, these data suggest that telomere recombination is likely involved in hTERT-mediated telomere shortening, and TPP1 might regulate the hTERT loading on telomeres for telomere recombination during the hTERT-mediated telomere-shortening process.

hTERT-mediated telomere shortening does not induce telomere signal-free ends and telomere dysfunction

We found that overexpression of hTERT did not increase the number of telomere signal-free chromosomal ends by T-FISH analysis, nor did it induce telomere dysfunctional foci formation (Fig. S5), which is consistent with previous reports.^24,25 In fact, expression of hTERT reduced the number of telomere signal-free ends in all the cell lines tested. Our observations are in agreement with previous reports that overexpression of hTERT in human primary cells leads to continued cell growth and extended life span in the absence of a DNA damage response.^1,27-29 These data suggest that hTERT-mediated telomere shortening does not produce telomere-free chromosomal ends and is different from aberrant rapid telomere deletion, which triggers a DNA damage response and ultimately initiates cell cycle arrest.

Cell culture, western blot analyses, and immunoprecipitation assays

ALT cell lines U2OS, SAOS-2, WI-35-VA13, and non-ALT cell lines MCF7, HT1080, IMR90, and WI38 cells were cultured in respective mediums (Clontech). Western blotting analyses and immunoprecipitation were essentially as described previously.⁵⁷

Ectopical expression of hTERT, lentiviral shRNA constructs, and antibodies

The lentiviral HA-tagged hTERT construct was transfected into 293T cells. After 48 h, supernatant media with virus were collected. When an equal titer of supernatant from hTERT construct or a vector control was transduced into different cell lines, almost all the cells became puromycin resistant, showing high transduction efficiency in the experimental system. After 48 h of infection with hTERT vector, hTERT mRNA and protein expressions were examined.

hTERT and TPP1 shRNAs were designed and cloned to the lentiviral vector (Sigma) (shRNA sequences are available upon request). The following antibodies were used: anti-mouse HA (abCAM) and anti-rabbit Flag (Sigma).

Measurement of chromosome-specific telomere length by fluorescent in situ hybridization

Chromosome arm-specific telomere length was measured by telomere quantitative fluorescent in situ hybridization (TQ-FISH) as previously described.¹⁴ Briefly, chromosome preparations were dropped onto clean microscopic slides and hybridized with 15 μl of hybridization mixture consisting of 0.3 μg/ml Cy3-labeled telomere-specific peptide nucleic acid (PNA) probe, 1 μl of co*cktails of FITC-labeled centromeric PNA probes specific for chromosomes 2, 4, 8, 9, 13, 15, 18, 20, and 21, and 20 μg/ml of Cy3-labeled centromeric PNA probes specific for chromosome X (Biomarkers), in hybridization buffer containing 50% formamide, 10 mM TRIS-HCl, pH 7.5, and 5% blocking reagent. Slides were denatured and then hybridized at 30 °C for 3 h. After hybridization, the slides were sequentially washed 10 min each at 42 °C: once in 1 × SSC, once in 0.5 × SSC, and once in 0.1 × SSC. The slides were then mounted in anti-fade mounting medium containing 300 ng/ml DAPI.

After TQ-FISH, cells were analyzed using an epifluorescence microscope equipped with a charge-coupled device (CCD) camera. Metaphase cells were captured with exposure times of 0.15, 0.25, and 0.05 s for Cy3, FITC, and DAPI signals, respectively. Digitized metaphase images were analyzed using the Isis software (MetaSystems Inc), which permits the measurement of 92 telomere signals simultaneously after karyotyping. Telomere fluorescent intensity units (FIU) were recorded as an indirect measurement of telomere length. For each study subject, 30 metaphase cells were analyzed.

Immunofluorescence and in combination with telomeric DNA-FISH assays

Cells were cultured on 4-well chamber slides, and immunofluorescence staining was performed after pre-extract in 0.5% Triton/PBS for 1 min on ice, as described previously.⁵⁸ To combine with telomeric DNA-FISH, after immunostaining, slides were fixed, treated with 0.5 μg/ml RNase A in 2 × SSC for 45 min at 37 °C, and then dehydrated in 70%, 80%, and 100% ethanol. After being denatured at 80 °C for 3 min, slides were hybridized for 2 h in hybridization solution contained 0.3 μg/ml Tel-C PNA telomere probe (Applied Biosystems) with 70% formamide, 1% BSA, and 10 mM Tris, pH 7.2. Finally, slides were washed, dehydrated, and covered with anti-fading 4’-6-diamidino-2-phenylindole (DAPI) mounting medium.

2D gel assays and TRF assays

Genomic DNA was isolated following standard procedure⁵⁹ and digested with Hinf I. Samples were separated by electrophoresis on 0.5% agarose gel at 1 v/cm for 18–20 h. For the TRF assay, alkaline transfer was performed. For the 2D gel assay, the separated DNA samples were cut from the gel and put into 1% agarose gel and continued to run for the secondary dimension at 5 v/cm for 2.5 h. Then the alkaline transfer was conducted. Membranes were hybridized with ³²P-labeled telomere probe.

TPP1-hTERT in vitro DNA binding assay

Buffers used and binding procedures were described previously.⁶⁰ Briefly, telomeric DNA substrates were biotinylated with a kit from BioServer Biotechnologies. In vitro-translated HA-hTERT (2 µL; Promega) and the biotinylated DNA substrates (0.1 pmol) were incubated in the absence or presence of various amounts of recombinant Flag-TPP1 for 30 min. The biotinylated DNA substrates were retrieved with streptavidin beads. After the unbound proteins were washed away with the binding buffer, HA-hTERT bound to the biotinylated DNA was eluted and detected by western blotting using anti-HA antibodies.

Additional material

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