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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: Mol Cancer Res. 2008 Oct;6(10):1582–1593. doi: 10.1158/1541-7786.MCR-08-0070

Suppression of hPOT1 in diploid human cells results in an hTERT dependant alteration of telomere length dynamics

Richard Possemato 1, Jamie C Timmons 1, Erica L Bauerlein 1, Naoya Wada 1, Amy Baldwin 3, Kenkichi Masutomi 4, William C Hahn 1,2
PMCID: PMC2741145  NIHMSID: NIHMS72246  PMID: 18922974

Abstract

POT1 is a 3′ telomeric single stranded overhang binding protein that has been implicated in chromosome end protection, the regulation of telomerase function and defining the 5′ chromosome terminus. In human cancer cells that exhibit constitutive hTERT activity, hPOT1 exerts control over telomere length. Primary human fibroblasts express low levels of catalytically active hTERT in an S-phase restricted manner that fails to counteract telomere attrition with cell division. Here we show that diploid human fibroblasts in which hPOT1 expression has been suppressed harbor telomeres that are longer than control cells. This difference in telomere length delays the onset of replicative senescence and is dependent upon S-phase restricted hTERT expression. These findings are consistent with the view that hPOT1 promotes a non-extendable telomere state resistant to extension by S-phase restricted telomerase. Manipulating this function of hPOT1 may thus hasten the cytotoxic effects of telomerase inhibition.

Keywords: Telomere, POT1, telomerase, senescence

Introduction

Telomeres are nucleoprotein complexes that cap the termini of eukaryotic chromosomes (1). Mammalian telomeres contain several thousand base pairs of TTAGGG repeats and end in a several hundred base pair 3′ extension of the G-rich strand that is required for the formation of a t-loop structure (2). Telomerase is a ribonucleoprotein reverse transcriptase composed of a catalytic protein subunit, TERT, and an RNA subunit, TERC, which together synthesize additional telomeric repeats (3–5). Dyskerin, an RNA binding molecule that participates in ribonucleoprotein assembly, is associated with catalytically active telomerase (6) and several other proteins have been reported to be associated with telomerase (7). In normal human cells, telomerase activity is tightly regulated. Although TERC is expressed constitutively in most cells, hTERT expression is restricted to low levels in S-phase of the cell cycle in most diploid non-malignant cells (8). This low level hTERT expression fails to maintain telomere length in such cells, and approximately 30–100 bp of telomeric DNA is lost per cell division (9). However, it is unclear whether this inability to maintain telomere length is because this low level of hTERT is not competent to act on telomeres or because of a suppressive mechanism inhibiting telomerase action at telomeres in these cells.

Normal human cells reach a replicative limit characterized by entrance into a growth arrested state termed replicative senescence, which can be triggered in part by dysfunctional telomeres (10). In these cells, entry into replicative senescence requires intact p53 or pRB cell cycle checkpoints (11, 12). In contrast, most immortalized and transformed cells exhibit constitutively high levels of telomerase activity, which correlates with stable telomere length (3, 13). This observation suggests that telomerase activation in pre-malignant cells results in an immortalized state that is required for tumorigenesis. In support of this hypothesis, over-expression of hTERT facilitates cell immortalization (14), while inhibition of telomerase either genetically or pharmacologically in already immortal cancer cell lines results in loss of telomeric sequences, a loss of proliferative capacity, and the onset of crisis (15–17). Consistent with the view that telomerase activation plays an important role in facilitating human cell transformation, co-expression of hTERT with cooperating oncogenes such as the simian virus 40 large T and small t antigens and RAS generates tumorigenic cells (18).

Telomere homeostasis is regulated by a multi-protein complex known as the telosome/shelterin complex (19). The results from studies focused on the control of telomere length by telomere binding proteins suggest that that such proteins make up a binary switch that controls whether a telomere is in an extendible or non-extendible state (20). In their initial studies in S. pombe, Baumann and colleagues identified POT1 as a novel telomere binding protein and a component necessary for the maintenance of a stable telomere (21). Subsequent genetic and biochemical studies have identified a role for POT1 in telomere length maintenance and telomere capping (22). Over-expression of full length or alternatively spliced hPOT1 (23) or suppression of hPOT1 by shRNA (24, 25) in the telomerase positive cell line HT1080 led to telomere elongation, while over-expression of mutant forms of hPOT1 lacking the 5′ OB fold domains led to telomere elongation in HTC75/HT1080 cells (22, 26). Recently a POT1 interacting protein, TPP1, has been identified, which regulates localization of POT1 to the telomere (25, 26) and mediates telomere length control in concert with POT1 (27–29).

In primer extension assays, the ability of POT1 to regulate telomere addition to artificial substrates depended upon primer composition. Primers that can fold into telomerase inaccessible G-quadruplex structures were resolved by the addition of POT1 and led to an increase in extension from these primers (30). Alternatively, extension from shorter primers was inhibited by POT1 (31, 32), but elongation, when it occurred, did so with increased processivity (32).

Unlike humans, two very similar POT1 genes, POT1a and POT1b are present in mice (33). Two groups have reported that targeted deletion of POT1a results in embryonic lethality in the mouse; however, these groups found both telomere elongation (34) and no change in overall telomere length (35) after germline deletion of POT1a. In contrast, targeted deletion of POT1b fails to change overall telomere length, but results in mTERC independent 3′ overhang lengthening (35).

Despite these extensive studies into the biology of POT1, the role of hPOT1 in regulating telomerase and telomere length in primary human cells or in cells lacking hTERT is currently unknown. Here we explored the consequences of suppressing hPOT1 under conditions where hTERT was constitutively expressed, S-phase restricted or suppressed. We found that suppression of hPOT1 alters the dynamics of telomere length control in an hTERT-dependent manner. These observations have implications for the timing of the entry of these cells into replicative senescence.

Results

Telomere length dynamics of primary human fibroblasts

To understand the role of hPOT1 in telomere length control in primary human cells, we generated lentiviral vectors co-expressing a puromycin or neomycin selectable marker and a short hairpin RNA (shRNA) that targets all known hPOT1 splice variants (hPOT1sh #1 puro and hPOT1sh #1 neo) or a shRNA that targets an unrelated sequence (CONsh puro or CONsh neo) and introduced these shRNAs into mortal BJ human fibroblasts. Expression of either hPOTsh #1 puro or hPOT1sh #1 neo induced a 75% decrease in the expression of hPOT1 as assessed by quantitative RT-PCR or immunoblotting (Fig. 1A).

FIGURE 1.

FIGURE 1

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Effects of suppressing hPOT1 on telomere dynamics. A. hPOT1 mRNA and protein levels. mRNA levels were determined by quantitative RT-PCR (top) and hPOT1 protein levels by immunoblotting (bottom) in BJ cells expressing either an hPOT1 shRNA (hPOT1sh #1) or a control shRNA (CONsh) in lentiviral constructs containing either puromycin (puro) or neomycin (neo) selection markers. Mean ± SD are shown for triplicate experiments. Arrows indicate hPOT1 isoforms. B. TRF Southern blotting of mortal BJ cells with passage. DNA was collected at the indicated population doublings (PD) after infection with CONsh (−) or hPOT1sh #1 (+). DNA size markers in kb are shown on the left. C. TRF Southern blotting of BJ cells that overexpress hTERT with successive passage. DNA was collected at the indicated PD after infection with a retrovirus encoding the hTERT cDNA in cells previously infected with CONsh (−) or hPOT1sh #1 (+). Lanes are numbered 1–12 for reference. D. Densitometry of selected lanes. The relative intensity at regular TRF length intervals is shown and mean TRF lengths for the entire lane are indicated. The upper left graph compares panel B lanes 1 (black diamonds) and 11 (grey squares); the upper middle graph compares panel B lanes 2 (black diamonds) and 12 (grey squares); the upper right graph compares panel B lanes 11 (black diamonds) and 12 (grey squares); the lower left graph compares panel C lanes 1 (black diamonds) and 11 (grey squares); the lower middle graph compares panel C lanes 1 (black diamonds) and 2 (grey squares); the lower right graph compares panel C lanes 11 (black diamonds) and 12 (grey squares).

To determine if suppressing hPOT1 expression and overexpressing hTERT in diploid fibroblasts resulted in an increased rate of telomere lengthening, we then infected BJ cells expressing CONsh neo or hPOT1 sh#1 neo with a retrovirus encoding the hTERT cDNA (BJ CONsh + hTERT and BJ hPOT1sh #1 + hTERT). Genomic DNA was isolated from these four cell lines (BJ CONsh, BJ hPOT1sh #1, BJ CONsh + hTERT and BJ hPOT1sh #1 + hTERT) every two population doublings (PDs) following infection and analyzed by telomere restriction fragment (TRF) Southern blotting in order to determine mean TRF length. In consonance with prior reports (9, 14), the mean telomere length decreased by 41 bp/PD in BJ CONsh cells, while BJ CONsh + hTERT cells exhibited overall telomere lengthening of 101 bp/PD (Figs. 1B odd lanes & 1C odd lanes). We noted a uniform decrease in TRF length in the BJ CONsh cell population, characterized by an increase in the signal of shorter fragments (compare Fig. 1B lanes 1 and 11 & see 1D upper left). When hTERT was expressed constitutively at high levels, we observed that the shortest TRFs increased in size rather than a uniform lengthening of telomeres of all lengths (compare Fig. 1C lanes 1 and 11 & see 1D lower left) as has been described in murine cells (36).

Surprisingly, we observed an altered telomere shortening dynamic in BJ cells in which hPOT1 had been suppressed. Over the time course of this experiment we detected relatively little change in the abundance of shorter TRFs (compare Fig. 1B lanes 2 and 12 & see 1D upper middle). As a result, when compared to control cells, the rate of telomere shortening in BJ hPOT1sh #1 cells was reduced by 52% to 20 bp/PD (Fig. 1B). After 12 PDs, this difference in the rate of telomere attrition results in a 400 bp increase in mean TRF length between BJ hPOT1sh #1 and BJ CONsh cells (compare Fig. 1B lanes 11 and 12 & see 1D upper right). Specifically, BJ CONsh cells exhibited stronger signals for TRF fragments shorter than 7kb (Fig. 1D upper right).

We found that either suppressing hPOT1 or over-expressing hTERT resulted in a smaller proportion of shorter TRF fragments with a corresponding greater proportion of longer fragments (compare Fig. 1C lanes 1 and 11 for BJ CONsh +hTERT cells & 1B lanes 11 and 12 for BJ hPOT1sh #1 cells and see Fig. 1D lower right and upper left). Cells in which both hTERT was overexpressed and hPOT1 was suppressed exhibited a 400 bp difference in telomere length within 2 PD after introduction of hTERT compared to cells overexpressing hTERT alone (compare Fig. 1C lanes 1 and 2 & see 1D lower middle). Subsequent telomere elongation proceeded at the same rate in both populations and thus BJ hPOT1sh #1 + hTERT cells maintained an increased telomere length throughout the course of this experiment (compare Fig. 1C lanes 1 and 2 to lanes 11 and 12 & see 1D lower middle and lower right). Taken together, these observations suggest that constitutive expression of hTERT and suppression of hPOT1 likely affect telomere length and/or structure in different ways.

To address the possibility that the observed telomere elongation phenotype was due to an off-target effect of RNAi, we generated a full length hPOT1 cDNA that is resistant to hPOT1sh #1 by introducing five nucleotide substitutions that do not change the amino acid coding sequence (hPOT1R). We introduced hPOT1R or a control vector into BJ and BJ hTERT cells (BJ hPOT1R and BJ hTERT hPOT1R). Expression of hPOT1R resulted in hPOT1 over-expression which persisted after subsequent introduction of hPOT1sh #1, whereas the control vector permitted hPOT1 suppression after introduction of hPOT1sh #1 (Fig. 2A). In BJ cells containing a control vector we again found a statistically significantly increase (p ≤ 0.000009) in TRF length of 230 bp upon introduction of hPOT1sh # 1. (Fig. 2B). In contrast, in BJ hPOT1R cells, we failed to observe telomere elongation upon introduction of hPOT1sh #1 (Figs. 2B & C), suggesting that the telomere elongation phenotype was not the result of off-target effects of RNAi.

FIGURE 2.

FIGURE 2

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Rescue of telomere length changes induced by hPOT1 suppression. A. Over-expression of hPOT1. hPOT1 mRNA levels in BJ or BJ hTERT cells expressing a control hairpin (CONsh) or an hPOT1 hairpin (hPOT1sh #1) in cells expressing the hPOT1sh #1 resistant version of hPOT1 (hPOT1R). B. TRF Southern blotting demonstrating rescue of hPOT1 suppression. The mean TRF length for each sample taken 10 PD after infection is indicated beneath the corresponding lane. p values (two-tailed t test) are shown for the indicated pair-wise comparisons from 3 (BJ) or 4 (BJ hTERT) replicate experiments. DNA size markers in kb shown on the left are for both gels. C. Quantitative telomere FISH demonstrating rescue of hPOT1 suppression. Results are shown for 12 metaphases for the BJ (left) and BJ hTERT (right) cell lines characterized in panels A and B. The fluorescence intensity of telomeres from each sample was assigned into a bin the intensity of which is indicated on the X-axis, and the number of telomeres contained within that bin indicated on the Y-axis. The percent increase in mean fluorescence intensity for the hPOT1sh #1 versus CONsh cell line is indicated. Fluorescence values are arbitrary and cannot be compared between graphs.

To determine if suppressing hPOT1 expression in diploid human fibroblasts that overexpress hTERT induced increased telomere length as has been observed in other telomerase positive human cell lines (24, 25), we suppressed hPOT1 in BJ hTERT cells, and also observed a statistically significant increase (p ≤ 0.000032) in telomere length that was abolished in cells co-expressing hPOT1R (Figs. 2B & C). The BJ hTERT fibroblasts used in these experiments had been passaged until they had achieved a stable maximum telomere length, allowing us to measure the effects of hPOT1 suppression on this maximal telomere length set point. Suppression of hPOT1 in the hTERT-immortalized BJ cells resulted in telomere elongation of approximately 340 bp beyond the stable maximum length as assessed by TRF Southern blotting (Fig. 2B).

Although the observed differences in telomere length are statistically significant, the differences in telomere length being detected in these experiments are at the limit of sensitivity for TRF Southern Blotting. Moreover, the estimation of size of the longest telomere lengths is less precise than that of shorter lengths. To confirm these measurements using a second approach, we employed quantitative fluorescence in situ hybridization (QFISH). This method does not measure sub-telomeric repeats, which comprise a much greater fraction of the TRF signal in primary cells (37), and therefore differences in telomere lengths as a percentage of overall telomere length is expected to be larger using this technique. As expected, when comparing primary BJ cells expressing CONsh to those expressing hPOT1sh #1, we noted an increase in fluorescence of 29%, which was abolished in cells expressing hPOT1R (Fig. 2C). Similarly, when comparing BJ hTERT cells to those expressing hPOT1sh #1, we noted an increase in fluorescence of 5.8%, which was also abolished by expression of hPOT1R (Fig. 2C). Together, these observations demonstrate that suppressing hPOT1 in both mortal and immortal human fibroblasts leads to telomere elongation and telomere maintenance at a new set point.

Effects of replicative history on the response of fibroblasts to hPOT1 suppression

We found that the introduction of two different hPOT1 shRNAs into early passage BJ and BJ hTERT cells increased the short term doubling time of the cells by 10–20% (Fig. 3A). A previous report showed that suppression of hPOT1 had no effect on the doubling time of immortal fibroblasts but increased the short term doubling time of non-immortal human fibroblasts by ~30–40% (38). In contrast, others have shown more dramatic effects of hPOT1 suppression on proliferative rates (24, 39). We speculated that the replicative history of the cells dictated their response to suppression of hPOT1.

FIGURE 3.

FIGURE 3

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Short-term effects of hPOT1 suppression in cells of different proliferative age. A. Doubling times of BJ and HFF cells. BJ or human foreskin fibroblast (HFF) cells infected with a control hairpin (CONsh) or one of two hairpins targeting hPOT1 (hPOT1sh #1 or #2) at the cumulative population doubling (PD) indicated. Doubling times were measured over 13–17 days in triplicate. B. hPOT1 mRNA levels in BJ and HFF cell at the indicated PD. Expression of hPOT1 for cell lines characterized in panel A using primers near the hPOT1sh #1 (top) or #2 (bottom) targeting site. C. SA β-gal staining. Values represent the percentage of cells that stained for SA β-gal. For these experiments, at least 100 total cells were scored in triplicate. D. TIF images. Staining for γ-H2AX (green) and telomeric DNA (red) are shown from two cells expressing hPOT1sh #1 that exhibited significant TIF formation, and two cells expressing CONsh that exhibited little to no TIF formation. E. Quantification of TIF formation. Values represent the percentage of nuclei containing >4 foci co-staining for γ-H2AX by indirect immunofluorescence and telomeric DNA by PNA FISH. At least 100 nuclei were scored in three independent experiments. F. DNA bridge formation. DAPI stained image of a representative DNA bridge (arrows) between two nuclei from PD 54 cells expressing hPOT1sh #1. DNA bridges were identified in 4% (8/200) of nuclei scored. DAPI signal has been pseudocolored white.

To test this hypothesis, we obtained freshly isolated human foreskin fibroblasts (HFFs) or used the aforementioned BJ fibroblasts. After allowing the cultures to proliferate for 0–40 additional PD, we introduced one of two shRNAs targeting all known splice variants of hPOT1 (hPOT1sh #1 or #2) or a control sequence (CONsh). hPOT1 was suppressed to similar levels in each of these cell populations, although we noted diminished hPOT1 expression in HFF cells that had undergone 40 PDs or BJ cells that had undergone 54 PDs (Fig. 3B). Suppression of hPOT1 in the HFF and BJ cells that had proliferated for the longest time in culture resulted in a 2–4 fold greater increase in doubling time, as well as a marked induction in SA β-gal staining (Figs. 3A & C and Supplemental Figure 1).

Prior work has shown that suppression of hPOT1 resulted in the appearance of telomere dysfunction induced foci (TIFs) (38). In order to determine whether TIFs were present in cells in which hPOT1 has been suppressed, we co-stained the early and late passage BJ cells described above for S15 phosphorylated histone H2AX (γ-H2AX) as a marker of DNA damage and a labeled PNA telomeric probe. We found co-localization of γ-H2AX and telomeric foci in both early and late passage cells in which hPOT1 was suppressed (Figure 3D & E). Although we observed γ-H2AX staining in control and hPOT1 suppressed cells, little to no co-localization with telomeres was detected in cells expressing the control shRNA (Figure 3D & E). The percentage of cells exhibiting γ-H2AX and telomeric focus co-localization was similar in early and late passage BJ cells. However, we also observed DNA bridges in late passage cells that lack hPOT1 expression at a frequency of 4% (8 nuclei out of 200, Figure 3D & F). Taken together, these observations suggest that suppression of hPOT1 results in telomere dysfunction in all cells regardless of passaging history. However, early passage cells are able to tolerate this damage while in later passage cells, suppression of hPOT1 results in telomere uncapping and the onset of senescence.

Long term effects of suppressing hPOT1 on telomere length and proliferative lifespan

With successive cell divisions, mortal human fibroblasts lose telomere length at the rate of approximately 30–50 bp per population doubling (9). Since cells in which hPOT1 was suppressed exhibited diminished telomere shortening, we assessed the effects of suppressing hPOT1 on telomere lengths over extended passage and on replicative lifespan. We cultured HFF and BJ cells expressing CONsh or hPOT1sh #1 or #2 and determined telomere length as a function of PD. Although cells in which hPOT1 was suppressed exhibited a longer mean telomere length over time relative to control cells, both populations eventually showed a reduction in mean telomere length at a similar rate (Figs. 4A & B). This observation contrasts with the decrease in telomere shortening rate seen immediately after suppression of hPOT1, suggesting that cells in which hPOT1 has been suppressed reach a new stable telomere length set point after which normal telomere attrition occurs.

FIGURE 4.

FIGURE 4

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Long-term effects of hPOT1 suppression on telomere length and replicative senescence. A. TRF Southern of BJ cells over a span of 34 PD. BJ cells were infected with a control hairpin (−) or an hPOT1 hairpin (+) and DNA collected at the indicated mean population doubling after infection. BJ cells used in this experiment are of a lower initial starting PD from those in Figs. 1 & 2. The mean TRF length for each sample is indicated beneath the corresponding lane. DNA size markers in kb are shown on the left. B. TRF Southern of HFF cells over a span of 30 PD. HFF cells were infected with a control hairpin (c) or one of two hPOT1-specific hairpins (#1 or #2) and DNA collected at the indicated mean population doubling after infection. The mean TRF length for each sample is indicated beneath the corresponding lane. DNA size markers in kb are shown on the left. C. Long-term proliferation of BJ hTERT cells after hPOT1 suppression. BJ hTERT cells were infected with a control hairpin (CONsh) or one of two hPOT1 hairpins (hPOT1 #1 or #2). Cumulative PD measurements represent an average of three separate cultures. D. Long-term proliferation of BJ cells after hPOT1 suppression. BJ cells were infected with CONsh or hPOT1 #1 or #2. Cumulative PD measurements represent an average of three separate cultures and the p value (two-tailed t test) indicates the degree of statistical significance between the pair-wise comparison of CONsh and either hPOT1sh #1 or #2 (p ≤ 0.03 for both comparisons). E. SA β-gal staining. Values represent the percentage of positively staining cells of at least 100 total cells scored in triplicate of cells from d 69 of the proliferation experiment shown in panel D.

To assess the effects of hPOT1 suppression in human fibroblasts on replicative senescence, we assessed the long term proliferative capacity of BJ or BJ hTERT fibroblasts expressing CONsh or hPOT1sh #1 or #2. Suppression of hPOT1 in immortal BJ fibroblasts did not affect proliferative capacity over the course of 100 d (Fig. 4C). However, suppression of hPOT1 in mortal BJ fibroblasts led to a statistically significant extension of the number of PD required to reach senescence (10 PD, Fig. 4D). This increase in proliferative capacity correlated with the predicted additional PD possible due to the additional telomere length found in cells in which hPOT1 was suppressed. Consistent with this observation, control cells exhibited a 2 fold increase in SA β-gal staining at an intermediate timepoint (Fig. 4E). Together, these observations indicated that there is a dichotomous effect of hPOT1 suppression on human fibroblasts: acute induction of the senescent state in later passage cells, and tolerance and extension in the time to replicative senescence in earlier passage cells.

Dependence of the telomere length effects of hPOT1 suppression on hTERT expression

To determine whether the telomere length phenotype seen in hPOT1 suppressed cells is dependent upon the presence of S-phase restricted hTERT, we generated a series of BJ and HFF cell lines in which we suppressed hTERT expression by introducing two different hTERT-specific shRNAs (hTERTsh #1 and #2). In BJ cells, we also over-expressed a catalytically inactive mutant of hTERT (DN-hTERT), which we had previously shown inhibits telomerase activity (15). We confirmed suppression of hTERT expression in these cells by RT-PCR and expression of DN-hTERT by immunoblotting (Fig. 5A). We note that suppression of hTERT led to an increase in hPOT1 expression in BJ, but not in HFF cells (Fig. 5B). Despite this change in hPOT1 levels, expression of hPOT1sh #1 in these cell lines was able to suppress hPOT1 to similar levels (Fig. 5B).

FIGURE 5.

FIGURE 5

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Effects of telomerase inhibition on hPOT1 dependent telomere lengthening. A. Inhibition of telomerase. BJ cells expressing a control hairpin (−) or a hairpin targeting hPOT1 (+) and either a second control hairpin (CONsh), one of two hairpins targeting hTERT (sh #1 or #2) or a dominant negative hTERT cDNA (DN) were analyzed by RT-PCR on RNA from cells synchronized in S-phase by serum starvation and release (COnsh, sh #1 and #2) or western blotting (CONsh and DN). B. hPOT1 mRNA levels. Relative hPOT1 expression was determined by quantitative RT-PCR on the cell lines described in panel A as well as HFF cells lines expressing a control hairpin (−) or a hairpin targeting hPOT1 (+) as well as CONsh or hTERTsh #1 or #2. C. TRF Southern of cells characterized in panels A and B. The mean TRF length at 14 population doublings after infection for each sample is indicated beneath the corresponding lane. Bar graphs show the change in telomere length after suppression of hPOT1 in the cell lines indicated. DNA size markers in kb are shown. D. Quantitative telomere FISH. Results of 12 metaphases for the BJ cell lines characterized in panels A–C are shown. The fluorescence intensity of telomeres from each sample was assigned into a bin the intensity of which is indicated on the X-axis, and the number of telomeres contained within that bin indicated on the Y-axis. The percent increase in mean fluorescence intensity for the hPOT1sh #1 versus CONsh for each pair of cell lines is indicated. Fluorescence values are arbitrary and cannot be compared between graphs.

Analysis of telomere length in both BJ and HFF cells demonstrated that suppression of hTERT by shRNA or inhibition of telomerase by DN-hTERT diminished the increase in telomere length seen after hPOT1sh #1 introduction by 50–100% as assessed by TRF length Southern blotting (Fig. 5C), and correlate with results obtained in BJ cells using QFISH (Fig. 5D). These observations indicate that the telomere elongation observed after hPOT1 suppression depends upon the presence of telomerase.

Discussion

Telomere length control by hPOT1

Prior reports have described the effects of perturbing POT1 function on telomere length in murine, galline and immortal human cells with constitutive telomerase expression (22, 25, 34, 35, 40). Here we investigated the effects of suppressing hPOT1 in normal human cells where telomerase activity is tightly regulated and telomeric DNA is lost with each round of cell division, a situation that represents normal human cells. We had previously demonstrated that hTERT is expressed in normal human fibroblasts in an S-phase restricted manner, but that this S-phase regulated telomerase does not affect overall telomere length (8). Therefore, it is unclear whether the inability of hTERT to maintain telomere length is because this low level of hTERT is not competent to act on telomeres or because of a suppressive mechanism inhibiting telomerase in these cells. Studies in S. cerevisiae further suggest that telomere elongation by telomerase is dependent upon telomere length and binding to Rif1 and Rif2 (20) or binding to Rap1p (41). These observations have led to the view that S-phase telomerase is restricted from elongating telomeres in mortal human cells due to a repressive mechanism located at the telomere.

Here, we present evidence that suppression of hPOT1 expression by shRNA in mortal diploid human cells altered normal telomere attrition resulting in a larger mean telomere length in cells in which hPOT1 has been suppressed. This change in telomere attrition occurred shortly after hPOT1 suppression resulting in telomere maintenance at a new set point. Once this new set point was reached, telomere attrition continued to occur at a normal rate with subsequent successive cell divisions.

We and others (22, 23, 25) have observed that suppression of hPOT1 in immortal cells that express telomerase constitutively leads to an increase in telomere length beyond the normally maintained set point. Here, we find that simultaneous overexpression of hTERT and suppression of hPOT1 in mortal BJ cells results in the combined effect of telomere elongation expected after hTERT overexpression in primary cells and the additional increase in telomere length noted after hPOT1 suppression. However, we noted neither a synergistic nor epistatic relationship between hTERT overexpression and hPOT1 suppression, suggesting that these manipulations may affect telomere length and/or structure in separate, non-complementary ways.

In normal human fibroblasts that express low levels of hTERT in S-phase, we observed that the increase in mean telomere length seen after hPOT1 suppression can be blunted by suppression of hTERT or inhibition of telomerase. These observations suggest that the S-phase restricted hTERT present in normal human cells is competent to act upon the telomere, but is inhibited by an hPOT1 dependent suppressive mechanism. However, since we were unable to completely eliminate the telomere elongation phenotype seen in these cells by suppressing hTERT expression, it remains possible that hPOT1 also participates in hTERT independent telomere replication. Indeed, an increase in homologous recombination has been observed upon deletion of POT1a in mice (33), and this increased recombination activity may also help explain the telomere lengthening observed here.

While this study is unique in its analysis of the effects of suppression of hPOT1 on telomere length and replicative senescence in primary human cells and the dependence of these effects on hTERT, our studies are consistent with prior studies in human, murine and galline cells that constitutively express hTERT. Suppression of POT1 in telomerase positive human (24, 25, 42) or galline cells (40) induces telomere elongation, and over-expression of an hPOT1 mutant lacking the 5′ OB fold domains leads to telomere elongation in immortal human cells (22, 26). However, others have found that hPOT1 over-expression in human cells that constitutively express hTERT can promote telomere elongation (23). Furthermore, in mice two groups have found both telomere elongation (34) or no change in overall telomere length (35) after germline deletion of POT1a, while targeted deletion of POT1b results in mTERC independent 3′ overhang lengthening (35). These manuscripts represent a growing body of literature supporting the notion that POT1 functions in telomere 3′ overhang protection, telomeric DNA damage signaling and cell cycle progression. However, because many of the conclusions regarding POT1 function vary depending on the model system used, we believe that studies in primary human cells are well positioned for understanding the role of hPOT1 in normal human biology.

Contributions to replicative and stress induced senescence

Human cells that undergo extensive passage in vitro eventually enter a metabolically active yet growth arrested state termed replicative senescence (43). In normal human cells, telomere attrition occurs with serial passage (9), and has been suggested to trigger the onset of replicative senescence (11). We found that suppression of hPOT1 in early passage BJ fibroblasts resulted in a population of cells with a longer mean telomere length that correlates with an increase in the number of PD required to reach replicative senescence.

Human cells can also be induced to enter senescence acutely by exposure to stresses such as oncogenic Ras (44), hydrogen peroxide (45), and X-ray irradiation (46). Moreover, when human cells reach replicative senescence, DNA damage response factors accumulate at telomeres in telomere dysfunction induced foci (TIFs) (47, 48). Suppression of hPOT1 in human fibroblasts induces TIF formation acutely in both primary and hTERT over-expressing cells, but has been shown to induce ATR dependant senescence only in primary cells (38, 40, 42, 49, 50). Here we show that the impairment of cell proliferation seen with hPOT1 suppression correlates with the proliferative history of primary BJ and HFF cells. Indeed, suppression of hPOT1 only induces senescence acutely in fibroblasts that have undergone a considerable number of population doublings. These observations are consistent with recent reports implicating proliferative age and/or levels of p16, which rise with passage, as key factors in the competency of cells to undergo acute senescence programs (51, 52). Alternatively, it is also possible that changes in telomere structure occur with successive passage that alters the response to suppression of hPOT1. In this case, one might expect that late passage cells are more dependent on hPOT1 for telomere end protection and acutely enter senescence in its absence. Indeed, while we observed induction of TIFs upon hPOT1 suppression in both early and late passage cells, we detected DNA bridges only in later passage cells. These data suggest that early passage cells are able to tolerate or compensate for reduced hPOT1 expression while in later passage cells suppression of hPOT1 results in telomeric damage and the onset of senescence. However, given the number of stimuli that are acting upon any given cell, a combination of telomeric and non-telomeric stresses may contribute to the senescence phenotype seen here.

Regulation of the telomere and induction of senescence are emerging as key processes underlying our ability to understand the development of cancer and the process of aging. The recent identification of senescent cells in pre-malignant and malignant human tissue highlights the importance of understanding the contributions of senescence to the suppression or progression of the malignant state (53). Telomeric stability and proper control of telomere length are important both in determining whether a cell enters the senescent state and are fundamental functional acquisitions found in cancer.

The multiple effects of suppression of hPOT1 on telomere length control, cellular proliferation, induction of senescence and the timing of the onset of replicative senescence provide additional insight into the interplay between telomere biochemistry and the senescence program. One approach to target telomeres involves agents that act by stabilizing G-quadruplex structures (54). Telomeric G-quadruplexes introduce kinks in telomeric DNA inhibit telomere elongation; therefore stabilization of G-quaruplexes would be expected to limit cell growth (55, 56). Biochemical studies demonstrate that hPOT1 can enhance the ability of G-quadruplex telomeric DNA to act as a telomerase substrate in vitro (30). Taken together, these observations suggest that targeting hPOT1 has the potential to complement the effects of G-quadruplex chemotherapeutics. The observations made in this study provide insight into how such targeting of hPOT1 may affect normal human cells.

Materials and Methods

Cell culture, lentiviral and retroviral constructs

Culture conditions and retroviral infections were performed as described (18, 57, 58). Human foreskin fibroblasts (HFF) were isolated from human neonatal foreskin by dispase mediated separation of the dermis from the epidermis. HFF from passage 2 were labeled as population doubling (PD) 0. The following sequences were used in the previously described (58) lentiviral vector pLKO.1-puro: CONsh (5′-ggcccgcaagctgaccctgaagttcat-3′), hPOT1sh #1 (5′-cggagcatgatttcttctaaa-3′), hPOT1sh #2 (5′-taagtgtctgatcaagctaca-3′). The puromycin selection cassette was replaced with a neomycin selection cassette in specified vectors. Infections were performed at the minimal titer required to achieve ~100% infection efficiency to ensure that simultaneously infected cultures had undergone the same number of PD. Retroviral constructs encoding a control hairpin or hTERT specific hairpins (8) and the DN-hTERT (15) construct are as described. The hPOT1R cDNA construct was generated by PCR cloning from pET14-Pot1 (kindly provided by J. Lingner) into pBabe-neo utilizing primers introducing 5 silent mutations (in capitalized, bold letters) into this hPOT1 cDNA (hPOT1R F 5′-cggagcTAGCtttctGctaaaggtatgg-3′; hPOT1R R 5′-agaaaGCTAgctccgtccacttctgct-3′) as well as two other gene specific primers (hPOT1 BamHI F 5′-acaggatccatgtctttggttccagca-3′; hPOT1 SalI R 5′-gctaaagctgacggcaatattagatta-3′). The resulting cDNA was fully sequenced.

Immunoblotting and RT-PCR

Immunoblotting for hPOT1 and H2AY was performed by isolating nuclear extracts as follows: cells were scraped into PBS, pelleted (1000 X g, 5 min), resuspended in harvest buffer (10 mM HEPES pH 7.9, 50 mM NaCl, 0.5 M Sucrose, 0.1 mM EDTA, 0.5% Triton, Roche mini-complete protease inhibitor), and incubated on ice for 5 min. Nuclei were pelleted (1000 × g, 10 min), washed in buffer A (10mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA), pelleted again, and the pellet lysed in buffer C (10 mM HEPES pH 7.9, 500 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.1% NP40, Roche mini-complete protease inhibitor). This nuclear lysate was sonicated (10 s), protein concentration determined by Lowry assay (Bio-Rad), and subjected to electrophoresis (100 μg) and transferred to nitrocellulose (Hybond ECL, Amersham). Guanidine denaturation was performed as described (22) prior to hPOT1 (rabbit polyclonal antibody 978 kindly provided by T. de Lange) and H2AY (Upstate Biotechnologies) antibody incubation. Antibodies specific for hTERT (Epitomics) and actin (Sigma) were used as described by the suppliers. The following primer pairs were used for quantitative RT-PCR (qRT-PCR) and RT-PCR: hPOT1 #1 (5′-catacctcgcacttcaagca-3′; 5′-ccaagatggaaatggtgtcc-3′), hPOT1 #2 (5′-tatccgtggttggaatgctt-3′, 5′-gctcaaacagggaaggtgag-3′), GAPDH (5′-gaaggtgaaggtcggagtca-3′; 5′-gacaagcttcccgttctcag-3′) and hTERT (LT5 and LT6 (59)). qRT-PCR was performed in triplicate using SYBR green master mix (Applied Biosystems, ABI 7300). C(t) values were determined using ABI prism software that includes automatic background correction and threshold selection. RT-PCR for hTERT was performed as described (8). Briefly, cells were synchronized in S-phase by contact inhibition and serum starvation for 48–72 h before release into serum containing medium for 21 h (CONsh) or 18 h (hPOT1sh #1). RT-PCR was performed using a one step RT-PCR kit (Superscript III, Invitrogen).

Analysis of telomere length

Telomere fluorescence in-situ hybridization (FISH) was performed as described (60). Twelve metaphases from colcemid (Sigma) treated cultures were imaged, and compared samples were imaged in the same microscopy session using the same exposure time. Telomeres were identified and fluorescence intensity measured using CellProfiler software (61) (cellprofiler.org). Telomeres of the same relative fluorescence were binned and the number of telomeres in each bin reported. The percentage change was determined by comparing average telomere intensity for the two populations tested. Telomere restriction fragment (TRF) Southern blotting was performed as described (9) and mean TRF length was measured as described (62) using 0.5% agarose gels. The program Image J was used (http://rsb.info.nih.gov/ij/index.html) to quantify signal intensity of the images shown in which the blank area (~6 cm) above the gel was cropped. p values of replicate experiments were determined by Student’s paired two-sided t test using the increase in TRF length for the compared cell lines as input. Telomere lengthening and shortening rates are the slope of a linear regression of a plot of TRF length versus PD.

Proliferation and Senescence Associated -Galactosidase (SA β-Gal)

Proliferation assays were performed in triplicate using a Coulter Particle Counter every 5–10 d for rapidly dividing cultures and every 10–20 d for pre-senescent cultures. Population doublings were defined as [log2(Cells counted/Cells plated)]. p values were determined by Student’s paired two-sided t test. Doubling time was defined as [Days/cumulative PDs] over 13–17 d. SA β-Gal staining was performed as described (63). The proportion of blue cells was determined by counting at least 100 cells from each triplicate well at the indicated time point or 4 PD after selection.

Telomere Dysfunction Induced Foci

Co-staining of γ-H2AX and telomeres was performed as described (64) with slight modifications. Cells seeded onto #1.5 coverslips were permiabilized (0.5% Triton X-100, 20mM Hepes pH7.9, 50mM NaCl, 3mM MgCl2, 300 mM Sucrose) prior to fixation. Images were captured on a Nikon Eclipse E800 microscope using a RT Slider Spot camera and a 100X Plan Apo oil immersion objective.

Supplementary Material

Supplemental F. SUPPLEMENTAL FIGURE 1.

Senescence Associated β-gal staining. Representative microscope fields of senescence associated β-gal staining from data collected in Figure 3C. Cells are shown with phase contrast at 100X magnification.

Acknowledgments

R.P. was supported by a Howard Hughes Pre-doctoral Fellowship. This work was supported in part by R01 AG23145 from the U.S. NIH NIA and an award from the Johnson and Johnson Focused Giving Program.

We thank Richard Maser, Kwok-Kin Wong, Ronald DePinho, James DiCaprio, Curtis Harris, Hans Widlund, Karl Münger, Peter Sicinski, Joanne Brugge, Jesse Boehm, Supriya Jain and members of the Hahn lab and the Cichowski lab for reagents, equipment and technical assistance. We also thank Titia de Lange and Stephanie Blackwood for the hPOT1 978 antibody, Joaquim Lingner and Patrick Reichenbach for the pET14pot1 plasmid, and Utz Herbig for assistance with the TIF experiments.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental F. SUPPLEMENTAL FIGURE 1.

Senescence Associated β-gal staining. Representative microscope fields of senescence associated β-gal staining from data collected in Figure 3C. Cells are shown with phase contrast at 100X magnification.

NIHMS72246-supplement-Supplemental_F.jpg (764.3KB, jpg)

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