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Human Molecular Genetics, 2004, Vol. 13, No. 3 285-293
DOI: 10.1093/hmg/ddh032

Limited capacity of the nuclear matrix to bind telomere repeat binding factor TRF1 may restrict the proliferation of mortal human fibroblasts

Jun Okabe1, Akiko Eguchi1,3, Renu Wadhwa1, Randeep Rakwal2, Rumi Tsukinoki4, Takao Hayakawa5 and Mahito Nakanishi1,*

1Gene Function Research Center and 2Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, Ibaraki, 305-8562, Japan, 3Japan Society for the Promotion of Science, Chiyoda, Tokyo, 102-8471, Japan, 4Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan and 5National Institute of Health Science, Setagaya, Tokyo 158-8501, Japan

Received August 27, 2003; Revised November 19, 2003; Accepted December 2, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The maintenance of telomere integrity is essential for prolonged cell proliferation, and failure in this mechanism is a most consistent manifestation of cellular senescence. In this study, we investigated the role of telomere repeat binding factor (TRF1) in the proliferation of human fibroblasts. TRF1 expression is upregulated in a large variety of immortal human cells and supports de novo telomere formation in a dose-dependent manner. These observations suggest that the suppression of TRF1 might limit telomere maintenance and thus the life span of mortal cells. However, primary fibroblasts ectopically overexpressing TRF1 were unable to avoid senescence. On the other hand, exogenously expressed TRF1 in primary fibroblasts neither supported de novo telomere formation nor bound to the nuclear matrix as tightly as observed in immortal cells that show upregulated TRF1 expression. We present evidence suggesting that mortal human cells lack specific ligand(s) that anchor TRF1 to the nuclear matrix and that this contributes to their limited lifespan.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The telomere is recognized as a complex dynamic structure actively involved in cell functions, including proliferation and the stress response, and failure of these functions contributes to senescence and crisis in human cells (1). The telomere consists of a cis-acting DNA element and trans-acting proteinaceous factors (1). The cis-acting element of human chromosomes is a repetitive duplex DNA (TTAGGG)n (telomeric repeat, TR) with a short 3' overhang. Maintaining the TR above a critical length is essential for the integrity of the telomere. In normal human somatic cells, telomeres undergo attrition every time the cell divides due to the end-replication problem. Consequently, old cells that have divided more times have shorter telomeres than young cells. The programmed telomere dysfunction induced by the attrition of the TR is accepted as one of the most consistent manifestations of cellular senescence. In sharp contrast to normal somatic cells, normal germline cells, embryonic stem cells, in vitro immortalized cells and tumour-derived cells have mechanisms that maintain the integrity of their telomeres. This is achieved either by activation of a specialized reverse transcriptase enzyme, telomerase, or by a recombination-based mechanism called ‘alternate lengthening of the telomere’ (ALT) (2). Ectopic expression of telomerase immortalizes some kinds of primary human cells (3–5).

On the other hand, it has become evident that TR length is not an isolated factor determining the onset of cell senescence and crisis (6). Most established tumour-derived cells have TRs much shorter (1–3 kbp) than the critical length of the TR in mortal cells (7). Furthermore, the critical length of the TR in mortal human cells can be altered under special experimental conditions (8). These observations suggest that the structure of the telomere, rather than the TR length itself, determines the onset of cell senescence and crisis. They also suggest that, in addition to the TR and the TR lengthening mechanism, trans-acting factors play some critical role in maintaining the integrity of the human telomere (6).

TRF1 (9) and TRF2 (10,11) have been identified as trans-acting factors directly associated with the human TR. Although both these proteins recognize the duplex (TTAGGG)n and are bound to the TR as homo-dimers, they have quite different functions, revealed by in vitro and in vivo assays. TRF1 bent linear TR DNA (12) and caused the pairing of two TR DNA molecules (13) in vitro, and functions as a negative regulator of TR length in vivo (14). On the other hand, TRF2 is thought to be responsible for protecting the chromosome terminus by catalysing t-loop formation (15) and for protecting the telomere from end-to-end fusions (16). The precise roles of these TRFs in programmed telomere dysfunction in mortal human cells have not yet been clarified.

We have analysed the requirements for de novo telomere formation in human cells, based on the frequency of telomere seeding (TS), which we used as an index of de novo telomere formation (17). TS is a phenomenon by which chromosome breakage is repaired by an exogenous DNA fragment (telomere seed) to create a novel telomere (18). TS in human cells depends strictly on the duplex (TTAGGG)n at the terminus of the telomere seed (19). Because TRFs strictly require the duplex (TTAGGG)n as their target, whereas telomerase can utilize more diverse sequences as templates, the frequency of TS (e.g., efficiency of de novo telomere formation) is thought to reflect the activity of TRFs in maintaining telomere integrity (19). In a previous study, we found that the frequency of TS increased in proportion to TR length in the telomere seeds (17). This rule could be applied to both mortal and immortal cells and was independent of the mechanism of TR maintenance (telomerase or ALT) (17). Furthermore, the minimum TR length required for full induction of TS varied among cell lines: cells with longer endogenous TRs required longer synthetic TRs, and mortal cells required very long TRs at the termini of the telomere seeds (17). These findings further support the idea that the TS faithfully reflects the intracellular environment necessary for maintaining telomere integrity.

Our second important finding was that the frequency of TS is dependent on the amount of available TRF1 but not TRF2 (17). Exogenous TR, as short as 500 bp, could induce TS fully when large amounts of TRF1 (>=90 molecules per 1 kbp endogenous TR) were available in the cells (17). In contrast, relatively small amounts of TRF2 (approximately nine molecules per 1 kbp endogenous TR) were sufficient for the full induction of TS, consistent with the proposed function of TRF2 in catalysing t-loop formation (15). Based on these data, we hypothesized that the total mass of TRF1 bound to the TR and the total length of the TR at the chromosome end were cooperative determinants of telomere integrity (17).

TRF1 is expressed ubiquitously in various tissues and cells (9,11). However, we found by quantitative immunoblotting that the expression of TRF1 protein varies among different cells, apparently according to their capacity for proliferation (immortal cell lines>primary fibroblasts and endothelial cells>peripheral blood mononuclear cells) (17). This result further suggests that, as well as the suppression of telomerase activity, the limited availability of TRF1, together with the attrition of TR, restricts the capacity of mortal cells to proliferate. In this study, we investigated whether the exogenous expression of TRF1 can extend the lifespan of primary human fibroblasts.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Immortalization of primary human cells is accompanied by the up-regulation of TRF1
We found previously that the expression of TRF1 in immortal human cells is significantly higher (48.8–16.2x103 molecules per cell) than in proliferating primary human fibroblasts (8.5–4.7x103 molecules per cell) or in quiescent peripheral blood mononuclear cells (<2.3x103 molecules per cell) (17). This finding suggests that a decrease in TRF1 expression is involved in programmed telomere dysfunction by restricting the proliferative capacity of mortal cells. In this study, we examined the amount of TRF1 in whole-cell extracts prepared from human fibroblasts before and after immortalization by various agents ({gamma}-irradiation, viral or cellular genes). As shown in Figure 1, TRF1 was up-regulated in the immortal cells compared with their parental mortal cells, independently of the agents used for immortalization. Interestingly, the upregulation of TRF1 was also independent of telomerase activity and of TR length (Fig. 1), suggesting that TRF1 may function independently of the proposed role in telomerase-dependent TR length regulation in immortal cells (14).



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  		Figure 1. Expression of TRF1 in human fibroblasts before and after immortalization. Primary human fibroblasts were immortalized by various agents, and whole-cell extracts were prepared from each cell line as described in Materials and Methods. Cell extracts (60 µg) were subjected to SDS–PAGE, then analysed by protein staining and immunoblotting using anti-TRF1 antibody. Top, SDS–PAGE stained with Coomassie Brilliant Blue R250 used as loading controls. Molecular weight markers are shown on the left. Upper middle, TRF1 detected by immunoblot. Lower middle, telomerase activity determined by TRAP assay. Relative telomerase activity is defined with reference to the activity in HeLa-LT cells, deemed to be to equal 1.0. n.d., not detected. Bottom, telomere length analysis by DNA blotting. Mean lengths of the terminal restriction fragments are indicated on the top of each image. Lane 1, TIG3; lane 2, HeLa-LT; lane 3, normal TIG; lane 4, immortal derivative of TIG with overexpression of mortalin and loss of p16INK4A; lane 5, normal MRC5; lane 6, MRC/SV (MRC5 immortalized by SV40 infection); lane 7, normal KMS6; lane 8, KMST6 (KMS6 immortalized by 60C{gamma}-irradiation); lane 9, normal WI38; lane 10, WI38/VA13 (WI38 immortalized by SV40 infection). m, mortal cells; i, immortal cells.

 
Effects of ectopic expression of exogenous TRF1 on the proliferation of primary human fibroblasts
Because TRF1 overexpression is associated with cell immortalization, we examined whether the ectopic expression of exogenous TRF1 in mortal human fibroblasts increases their limited proliferative capacity. We obtained several cell clones stably expressing Flag-tagged TRF1 (Flag-TRF1) by transfecting a Flag-TRF1 expression vector into two different primary human fibroblast cell lines (TIG3 and TIG7). These cell clones could be divided into the following two categories by the level of Flag-TRF1 in the cell extracts: cells expressing Flag-TRF1 very strongly (25- to 50-fold excess of endogenous TRF1; Fig. 2A, lanes 5, 10 and 11) and cells expressing it moderately (5- to 15-fold excess; Fig. 2A, lanes 3, 4 and 12). We confirmed the uniform expression of Flag-TRF1 in these clonal populations by indirect immunofluorescence microscopy using anti-Flag antibody (data not shown). We then compared the proliferative capacity of these cloned cells with those of control clones obtained by transfecting cells with empty vector. As shown in Figure 2C, the cells expressing large amounts of Flag-TRF1 (closed circles) proliferated for five to 20 more population doublings (PDs) than the control cells (open circles) before they ceased to divide. On the other hand, the lifespans of cells expressing Flag-TRF1 moderately (grey circles) were similar to those of control cells.



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  		Figure 2. Proliferation, senescence phenotype, and telomere-repeat shortening of primary human fibroblasts supplemented with exogenous TRF1. (A and B) Quantitation of TRF1 in primary human fibroblasts transduced with exogenous TRF1. TRF1 in whole-cell extract was determined by immunoblotting, as in Figure 1. F-T, Flag-TRF1; T, native TRF1. (A) Analysis of cell clones transduced with Flag-TRF1 expression vector. Control vector (lanes 6, 7, 8, 13, 14 and 15), cell clones transduced with empty vector carrying drug-resistant gene (Neor). No vector (lanes 9 and 16), cells before gene transduction. TX36 (lane 2), HeLaLT cells expressing Flag-TRF1 (17). (B) Analysis of pooled cell culture transduced with Flag-TRF1 or native TRF1 genes by retroviral vectors. URJ1 (lanes 3–5) and URJ2 (lanes 12–14), TIG3 cells transduced with native TRF1; URK3 (lanes 6–8), TIG3 cells transduced with Flag-TRF1. All the other transduced pooled cultured cells expressed similar amounts of Flag-TRF1 or native TRF1 (data not shown). Vector (lanes 9–11), TIG3 cells infected with empty retroviral vector. UX244 and UX224, TIG3 cells stably expressing Flag-TRF1, as shown in (A). PDs, population doubling counts after retroviral infection. (C and D) Proliferation of primary human fibroblasts supplemented with exogenous TRF1. (C) Proliferation of cloned cell lines. Solid circles, cells expressing Flag-TRF1 strongly (UX244 derived from TIG3 cells; VX221 and VX222 derived from TIG7 cells, as shown in A); grey circles, cells expressing Flag-TRF1 moderately (UX224 and UX225 derived from TIG3 cells; VX242 derived from TIG7 cells); open circles, cells transduced with empty vector carrying drug-resistant gene (Neor). Day zero is defined as the first day upon which we counted the number of cells derived from each colony. (D) Proliferation of pooled cultures. Solid squares, cells transduced with native TRF1; solid circles, cells transduced with Flag-TRF1; open circles, cells infected with empty vector. Day zero is defined as the seventh day after retroviral infection. Inset in (D), late phase of proliferation of pooled cultures. Day zero is defined as the day upon which the cells had proliferated for 30 PDs (TIG3) or for 35 PDs (TIG7). (E and F) Characterization of the senescent phenotype in TIG3-derived pooled cultures transduced with retroviral vectors. (E) Observation by differential interference contrast microscopy. The cells were fixed and stained to detect SA-ß-galactosidase activity, as described in Materials and Methods. TRF1, the pooled URJ1 culture expressing exogenous TRF1 (shown in B); vector, the pooled culture transduced with empty vector. (F) Quantitation of cells expressing SA-ß-gal in the pooled cultures. The cells were stained and observed as in (E) and the percentage of total cells expressing SA-ß-gal in the view is indicated for each PD. Solid circle, the pooled URJ1 culture expressing exogenous TRF1; open circle, the pooled culture transduced with empty vector. (G and H) Telomere length analysis in TIG3-derived pooled cultures transduced with retroviral vectors. (G) DNA blot analysis. Genomic DNA (5 µg) was prepared from the pooled URJ1 culture (TRF1) or from TIG3 cells transduced with empty vector (vector) after cells had proliferated for the indicated PDs, as in (D). DNA was analysed by DNA blotting, as described in Materials and Methods. Molecular weight markers are shown on the left. (H) Rate of telomere shortening. Mean telomere length was calculated from the results shown in (G) as described in Materials and Methods.

 
The data described above suggest that TRF1 expressed ectopically partially overcame the proliferation limit of mortal fibroblasts if it was supplied in large excess. However, the results obtained from a limited number of cell lines might be biased by the characteristics of each cell clone, affecting the survival of cells under harsh selective conditions. Therefore, we re-examined this phenomenon using a pooled culture of primary human fibroblasts expressing either Flag-TRF1 or TRF1 after retrovirus-mediated gene transduction (Fig. 2D). Under the experimental conditions employed, 50–90% of the cells became resistant to blasticidin S after gene transduction, and more than 90% of these transduced cells stably expressed exogenous Flag-TRF1 or TRF1, at a 20- to 35-fold excess of the endogenous TRF1 (Fig. 2B). We found that some pooled cultures expressing exogenous TRF1 (3/6 of TIG3-derived cultures and 1/4 of TIG7-derived cultures) proliferated for four to six more PDs than the control cells, even under these non-biased conditions (Fig. 2D). Based on these results, we conclude that excess TRF1 might partially compensate for the limited proliferative capacity of primary human fibroblasts (four to 20 PDs depending on the experimental design), but cannot overcome their proliferative limit by itself.

Supporting this conclusion from another perspective is our finding that supplementation of exogenous TRF1 delayed, but did not prevent, the onset of the senescent phenotype (20) in primary human fibroblasts. When TIG3-derived pooled-cultured cells proliferated for 41 PDs after retrovirus-mediated gene transduction, 80% of the control cells expressed senescence-associated ß-galactosidase (SA-ß-gal), whereas the cells that acquired enhanced proliferation capacity from supplemented exogenous TRF1 expressed little SA-ß-gal (Fig. 2E and F). The control cells also had an enlarged and flattened morphology characteristic of senescent cells at 41 PDs, whereas the cells expressing exogenous TRF1 maintained the spindle-like shape characteristic of actively proliferating cells at this stage (Fig. 2E). However, the latter cells exhibited the characteristic senescence phenotypes at 45 PDs (Fig. 2F), indicating that excess TRF1 might delay but cannot prevent the onset of cell senescence. A similar delay in the onset of the senescence phenotype was observed in cloned cell lines stably expressing exogenous TRF1 (data not shown).

The effect of exogenous TRF1 on proliferation of primary human fibroblasts was apparently independent of the modulation of other cell phenotypes related to cell growth and to telomere integrity. Previously, Shen et al. (21) reported that the overexpression of Pin2 (a splicing variant of TRF1) in HeLa cells affected cell growth by arresting the cell cycle at G2/M phase. However, we found that the expression of exogenous TRF1 in primary human fibroblasts affected neither cell growth (Fig. 2D) nor the DNA synthesis index (data not shown), indicating that the phenomenon we observed did not result from an artificial delay in cell proliferation. TRF1 is also known to be a negative regulator of TR length (14), but exogenous TRF1 did not affect the rate of TR shortening in primary human fibroblasts under our experimental conditions (Fig. 2G and H), excluding the possibility that TRF1 affects cell proliferation indirectly by altering TR length.

Supplemented exogenous TRF1 does not support de novo telomere formation in primary human fibroblasts
As described above, TRF1 expressed ectopically in primary human fibroblasts extended their lifespan, but only partially, during cell senescence and crisis. Although this observation may indicate that TRF1 contributes little, if anything, to programmed telomere dysfunction, it is also possible that TRF1 expressed ectopically in mortal fibroblasts cannot express the function that is essential for prolonged cell proliferation for some reason. Therefore, we examined the biological function of exogenous TRF1 in primary human fibroblasts from another point of view.

We have demonstrated previously that TRF1 supports de novo telomere formation in a dose-dependent manner in immortal cells: sufficient supplementary TRF1 is required to induce TS fully, at a frequency of 90% (17). In contrast, TS is induced quite inefficiently in primary human fibroblasts (22) and requires a TR as long as 2 kbp at the terminus of the telomere seed for induction (17). Although the results observed in immortal cell lines clearly reveal the importance of the interaction between TRF1 and the TR in de novo telomere formation, the inefficient TS observed in mortal fibroblasts may indicate that TS requires the TR elongation mechanism in addition to an abundant supply of TRF1. Because the telomere seeds are linear DNA fragments containing relatively short TR (0.5–2 kbp) at their termini and because they lack the 3' single-stranded telomere overhang which is essential for stabilizing the chromosome ends through t-loop formation, it is reasonable to suppose that telomerase may be involved in de novo telomere formation by creating a functional cis DNA element with a long duplex (TTAGGG)n and an appropriate 3' overhang.

However, we maintain that the apparent lack of telomerase activity per se does not account for the inefficient TS observed in primary human fibroblasts, for the following reasons. Firstly, we found that the length of the TR present in the telomere newly established by TS was essentially the same as the length of the TR present in the telomere seed (17). This phenomenon was observed even in cells with long endogenous TR (e.g., HeLa-LT cells) and in primary human fibroblasts, which may require a TR of sufficient length to avoid premature senescence (e.g., TIG3 and TIG7 cells) (17). We determined the length of the TR in the newly established telomere after a single-cell clone divided for over 20 doublings to produce 106 cells or more. It is quite unlikely that elongation of the TR is required within a few days after drug selection, during which time the fate of the telomere seed was decided: either to establish a new telomere or to integrate as an internal part of the chromosome. Secondly, the mechanism for maintaining the 3' single-stranded telomere overhang acts in both immortal cells and primary human fibroblasts (23). The low but significant levels of telomerase activity detected in primary fibroblasts (including the TIG3 cells used in this study) is also thought to be partly responsible for maintaining the 3' single-stranded overhang (23). Therefore, as long as we used actively proliferating primary fibroblasts, we should expect that the mechanism that maintains the 3' single-stranded telomere overhang is active in the cells. For these reasons, we inferred that TS could be used as an index with which to estimate the biological function of TRF1 in terms to de novo telomere formation, even in mortal cells with only a trace of telomerase activity.

We then examined whether ectopically expressed TRF1 compensates for the inefficient TS in primary human fibroblasts, using telomere seeds with 2 kbp TRs. To our surprise, we found that supplementation of a 25-fold excess TRF1 did not affect the frequency of TS in primary human fibroblasts (22 versus 24%) (Table 1). This result contrasts with our previous finding that supplementation with excess TRF1 significantly increased the frequency of TS induced in HeLa cells by a telomere seed with a 0.5 kbp TR (36% without excess TRF1 versus 73–83% with excess TRF1) (17). From these observations, we conclude that exogenous TRF1 in mortal human fibroblasts lacks the activity required to maintain telomere integrity, even when present in a 25-fold excess relative to endogenous TRF1.


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  		Table 1. Effect of excess TRF1 on de novo telomere formation
 
To clarify why exogenous TRF1 has different effects on de novo telomere formation in mortal human fibroblasts and HeLa cells, we compared the biochemical properties of the TRF1 molecules recovered from these cells. Because phosphorylation (24) and poly(ADP-ribosyl)ation (25) of TRF1 modulate the biological activity of this protein (e.g., binding to duplex TR), we examined the mobility of TRF1 on two-dimensional gel electrophoresis (2-DE) (Fig. 3A and B). The Flag-TRF1 recovered from immortal TRK1 cells (pooled HeLa-LT cells expressing Flag-TRF1) and that recovered from mortal URK3 cells (pooled primary human fibroblasts expressing Flag-TRF1) both formed two distinct spots on 2-DE, with identical isoelectric points (pI=5.6 and 5.8), indicating that the differential biological activities of TRF1 protein in normal and transformed cells do not depend on this sort of modification.



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  		Figure 3. Biochemical characterization of Flag-TRF1 protein expressed in primary human fibroblasts and HeLa cells. (A and B) Two-dimensional gel electrophoresis (2-DE) analysis of nuclear extract prepared from cells expressing Flag-TRF1. (A) 2-DE immunoblot analysis. Nuclear extract was prepared from pooled cultured cells expressing Flag-TRF1 transduced by retroviral vector, then subjected to 2-DE and analysed by immunoblotting using anti-TRF1 antibody, as described in Materials and Methods. Molecular weight markers and pI markers are shown on the left and the top, respectively. TRK1, pooled HeLa-LT cells expressing Flag-TRF1; URK3, pooled TIG3 cells expressing Flag-TRF1. F-T, Flag-TRF1; T, endogenous TRF1. (B) Quantitation of Flag-TRF1 signals. The intensity of signals corresponding to Flag-TRF1 in (A) was quantitated with a fluorescence image analyser in the first dimension of 2-DE. pI markers are shown on the bottom. (C and D) Gel filtration analysis of nuclear extract prepared from cells expressing Flag-TRF1. (C) immunoblot analysis of fractionated extract. Nuclear extract was prepared, fractionated on a Sephacryl S300 HR column, and analysed by immunoblotting using anti-TRF1, as described in Materials and Methods. TRK1, URK3, F-T and T are defined as in (A). (D) Quantitation of Flag-TRF1 signals. Intensity of signals corresponding to Flag-TRF1 in C was quantitated as in (B), and is given as a percentage of total signal intensity. Molecular weight markers are shown on the bottom.

 
TRF1 is also reported to exist as a complex with other molecules. The reported size of the TRF1-containing complex varies significantly, from >1000 kDa (26) to 100–120 kDa (12), probably depending on the experimental conditions. We examined the size of the TRF1-containing complex using a Sephacryl S300 gel filtration column equilibrated with buffer B containing 0.5 M KCl and 10 mM CHAPS, which we used for extracting TRF1 from cells. Under these conditions, both of these Flag-TRF1 proteins migrated as a single complex of ~280 kDa (Fig. 3C and D). Therefore, it is unlikely that the difference in the biological activities of these TRF1 molecules is dependent on a drastic reorganization of the TRF1-containing complex. From these data, we conclude that the biochemical properties of the exogenous TRF1 protein in mortal and immortal cells are essentially identical and that the biological activity of TRF1 involved in maintaining telomere integrity is regulated by other mechanism(s).

Nuclear matrix of primary human fibroblasts has a limited capacity to bind TRF1
Association with the nuclear matrix is one of the characteristics of the human telomere, distinguishing it from other parts of the chromosome (27,28). TR-binding proteins are thought to mediate this association (28). TRF1 was detected as a component of the nuclear matrix (28) and is dissociated from the matrix only under high-salt conditions (26), suggesting that TRF1 might be responsible for anchoring the TR to the nuclear matrix. Exogenous Flag-TRF1 expressed excessively in HeLa cells with short TRs, in which the major fraction of TRF1 should exist free from TR, was also localized in the nuclear matrix (data not shown). This indicates that its association with the nuclear matrix is an intrinsic property of TRF1, independent of TR binding. TRF1 is a typical karyophilic protein with a nuclear localization signal and is predominantly localized in the nucleus, even when overexpressed in both primary human fibroblasts and HeLa cells by gene transduction (data not shown). During the course of characterizing the sub-cellular fractions containing TRF1, however, we found that exogenous Flag-TRF1 expressed in primary fibroblasts dissociated from the nuclear fraction more readily than exogenous Flag-TRF1 expressed in HeLa cells. When whole cells were extracted under low-salt conditions (<=0.2 M NaCl) in the presence of 0.5% Triton X-100 (29), only 20% of the Flag-TRF1 expressed in primary fibroblasts was recovered in the insoluble fractions (Fig. 4, middle right), whereas 65% of Flag-TRF1 was recovered in the insoluble fraction of HeLa cells under the same conditions (Fig. 4, upper right). In contrast, most of the endogenous TRF1 in primary fibroblasts and HeLa cells (80 and 70%, respectively) was recovered in the insoluble fraction under the same conditions (Fig. 4, middle left and upper left, respectively). This difference was not due to the artificial destruction or contamination of the nuclear fraction because vimentin and lamins were always recovered in the insoluble fraction (data not shown). Therefore, extraction under low-salt conditions is a unique property of exogenous TRF1 expressed ectopically in primary human fibroblasts, suggesting that the nuclear matrix of primary human fibroblasts has a limited capacity to bind TRF1.



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  		Figure 4. Biochemical fractionation of TRF1. Quantitation of TRF1 in stepwise salt extracts. Signals corresponding to either endogenous TRF1 or exogenous Flag-TRF1 on the immunoblot were quantitated and are given as a percentage of total signal intensity, as described in Materials and Methods. Left, quantitation of endogenous TRF1. Signals corresponding to endogenous TRF1 were analysed in immortal HeLa-LT cells (top); in mortal TIG3 cells (primary human fibroblast) (middle); and in TIM cells (an immortal derivative clone of TIG overexpressing mortalin and having lost p16INK4A) (bottom). Right, quantitation of exogenous Flag-TRF1. Signals corresponding to Flag-TRF1 were analysed in TRK1 cells (immortal HeLa-LT cells expressing Flag-TRF1) (top); in URK3 cells (mortal TIG3 cells expressing Flag-TRF1) (middle); and in IRK1 cells (immortal TIM cells expressing Flag-TRF1) (bottom).

 
Because HeLa cells originated from epithelial cells, which have different characteristics from fibroblasts in cell proliferation and in the induction of cell senescence, we examined the dissociation of endogenous TRF1 and exogenous Flag-TRF1 from the nuclei of TIG3-derived immortal fibroblasts (TIM cells, shown in Fig. 1) by stepwise salt extraction. In TIM cells, 95% of endogenous TRF1 (Fig. 4, lower left) and 90% of exogenous Flag-TRF1 (Fig. 4, lower right) remained associated with the fractions not extractable under low-salt conditions (<=0.2 M NaCl), suggesting that the capacity of the nuclear matrix to bind TRF1 is up-regulated during the immortalization of primary human fibroblasts.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Elucidation of the mechanism underlying the programmed dysfunction of human telomeres is critical for our understanding of the principles of ageing, tissue regeneration, and carcinogenesis. The TR is an important cis-acting element of the intact telomere and the maintenance of the TR at a sufficient length is essential for prolonged cell proliferation (1). However, accumulating evidence suggests that the TR is not an isolated determinant of telomere integrity. Considering that the rate of spontaneous immortalization of primary human fibroblasts is unusually low (<=10–7) (30,31), the maintenance of the mortal phenotype in differentiated human cells may involve multiple suppression mechanisms, including the suppression of telomerase activity (32). In this context, the role of TR-binding proteins has attracted attention.

In this and previous studies (17), we reported that the amount of TRF1 protein varies among cell lines and relates to the capacity of cells to proliferate (immortal cells>proliferating mortal cells>quiescent mortal cells). Up-regulation of TRF1 seems to be a general phenomenon associated with cell immortalization, independent of the agents triggering immortalization and of the mode of TR elongation (Fig. 1). In a previous study, we also identified TRF1 as a limiting factor in de novo telomere formation and concluded that the total mass of TRF1 bound to each TR was one of the determinants of telomere integrity (17). These results strongly suggest that the limited availability of TRF1 in primary human fibroblasts, as well as the suppression of telomerase activity, restricts their capacity to proliferate. In the present study, we show that supplementation of exogenous TRF1 extends the lifespan of primary human fibroblasts and delays the onset of cell senescence, for four to 20 PDs depending on the experimental design (Fig. 2). Although these data suggest that TRF1 is potentially involved in regulated telomere dysfunction, the expression of excess TRF1 is insufficient by itself to support the long-term proliferation of primary human fibroblasts.

On the other hand, we also found that exogenous TRF1 expressed ectopically in primary fibroblasts cannot support de novo telomere formation, even though the amount of TRF1 detected by protein blotting was sufficiently high (Table 1). The apparent lack of telomerase activity alone may not account for the inefficient TS induction in primary fibroblasts (see the discussion in the Results section). Therefore, this observation indicates that, in primary human fibroblasts, exogenous TRF1 cannot acquire its physiological function essential for the maintenance of telomere integrity. Because biochemical characterization did not reveal a difference between the exogenous TRF1 molecules expressed in mortal cells and those expressed in immortal cells (Fig. 3), this defect may not be caused by alternative properties of TRF1 but rather by a defect in other cellular factor(s) essential for TRF1 to exhibit its function as a trans-acting factor of the intact telomere.

TRF1 associates with the nucleus by an intrinsic activity distinct from TR binding, and majority of endogenous TRF1 dissociates from the nucleus only in the presence of high salt (>=0.3 M NaCl; Fig. 4, left). Our finding that exogenous TRF1 expressed ectopically in primary human fibroblasts dissociates from the nucleus more readily under low-salt conditions (<=0.2 M NaCl) than TRF1 expressed in immortal cells (HeLa cells and TIG3-derived TIM cells; Fig. 4, right) suggests that some specific ligand(s) anchoring TRF1 to the nuclear matrix through electrostatic interactions is suppressed (or inactivated) in mortal cells but is induced (or activated) in immortal cells. If a tight association with the nucleus is essential for TRF1 to function in the maintenance of telomere integrity, it is reasonable to assume that the exogenous TRF1 expressed ectopically in mortal human fibroblasts neither supports de novo telomere formation nor overcomes the proliferation limit of the cells.

Among a number of TRF1-binding proteins identified recently, such as TANK1 (25), TIN2 (33), NBS1 (34), Ku (35), PinX1 (36), SALL1 (37) and Pot1 (38), TANK1 could be a candidate for this hypothetical partner of TRF1 because this molecule is tightly associated with the nuclear membrane (25). However, the apparent lack of a nuclear localization signal on TANK1 and its complex localization in interphase nuclei (39) are inconsistent with the expected properties of this hypothetical molecule. Even if TANK1 is responsible for anchoring TRF1 to the nuclear matrix, its poly(ADP-ribosyl)ation activity may not be involved in this phenomenon, because the isoelectric points of TRF1 recovered from immortal cells and from mortal cells are identical.

Tight regulation of mortality in terminally differentiated human tissue cells is an important issue in maintaining the homeostasis of individual bodies by avoiding tumourigenesis, and the suppression of telomerase activity is responsible for maintaining the mortality of human cells. It has recently been reported that multiple signal-transduction pathways coordinately suppress the transcription of the telomerase catalytic subunit (TERT) gene (32) and that this complex system inhibits the accidental activation of telomerase in mortal cells (32). In this context, it is reasonable to infer that the lifespan of mortal human fibroblasts is circumscribed by the suppression of multiple independent mechanisms, including the suppression of telomerase and the suppression of the mechanism that maintains the 3' single-stranded telomere overhang (23,40). Our observation that both the expression of TRF1 and the capacity of the nuclear matrix to retain TRF1 are co-ordinately suppressed in primary human fibroblasts may provide clues to further mechanisms that ensure the tight regulation of cell mortality.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells
TIG3, TIG7, MRC5, KMS6 (human fetal primary fibroblasts) and KMST6 (60Co {gamma}-irradiated KMS6) cells were obtained from the Health Science Research Resource Bank (Tokyo, Japan). These cells were cultured as recommended by the suppliers. MRC/SV [simian virus 40 (SV40)-transformed MRC5] and TIM (an immortal derivative clone of TIG that overexpresses mortalin and has lost p16INK4A; Wadhwa, R. and Kaul, S.C., unpublished data) (41–43) were cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin and streptomycin (Invitrogen BV, Groningen, The Netherlands) for nearly 100 PDs. The other cells were cultured as described previously (17). Cells were passaged at a dilution of 1 : 4–16 when they reached 80% confluence.

Primary human fibroblasts expressing TRF1 ectopically were obtained either by transfecting Flag-tagged TRF1 (Flag-TRF1) expression vector, as described previously (17), or by retrovirus-mediated gene transfer as described below. To obtain retroviral vectors, cDNAs encoding either human TRF1 or Flag-TRF1 was inserted between the BamHI and NotI sites of pCX4bsr (44) (a kind gift from Dr T. Akagi, Osaka Bioscience Institute, Suita, Osaka, Japan) to create pJOJ50 and pJOJ62, respectively. Each of these plasmids was co-transfected with pCL-Ampho (45) into BOSC23 cells (46). Two days after transfection, culture supernatants were collected and used as viral stocks. TIG3 or TIG7 cells were seeded at 5x105 cells/dish in a 60 mm dish for 20 h before infection, and incubated with the virus stock for 2 days. The cells were then selected with blasticidin S (8 µg/ml; Invivogen, San Diego, CA, USA) for 7 days. The cells were examined by indirect immunofluorescence microscopy and by protein blotting, using the anti-Flag monoclonal antibody, M2 (Eastman-Kodak, Rochester, NY, USA), or the anti-human TRF1 rabbit serum, JE#1, as described previously (17).

For the cell proliferation assay (Fig. 2C), primary cell clones consisting of about 100 cells were carefully isolated using a cloning cylinder to avoid loss of cells. The cells were then passaged into a larger-scale culture after they had reached about 80% confluence. Cell number was determined manually after the total cell number reached about 106.

Biochemical characterization of TRF1
Nuclei were isolated as described previously (28). Samples for two-dimensional electrophoresis (2-DE; Fig. 3A and B) were prepared by extracting the isolated nuclei with buffer A [6 M urea, 1 M thiourea, 3% CHAPS, 0.5% 2-mercaptoethanol, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF and protease inhibitor cocktail (PIC: 1.7 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 10 µg/ml aprotinin, 10 µg/ml E-64)], as described previously (47). The isoelectric point (pI) of TRF1 was estimated using standard 2-DE markers (BioRad). Samples used to evaluate the size of the TRF1-containing complex (Fig. 3C and D) were prepared by extracting the nuclei with buffer B, previously described as buffer C (16), except that we substituted 0.2% NP40 with 10 mM CHAPS. Samples were examined on a Sephacryl S-300HR column (11x1000 mm; Amersham Biosciences, Bucks, UK) equilibrated with buffer B. Each fraction was examined by protein blotting after separation by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) (8.5%), as described previously (17), and the protein blotting signals were quantitated with a chemiluminescence image analyser (LAS1000 plus; Fuji, Japan). The size of the TRF1-containing complex was estimated using a Gel Filtration Calibration kit (Amersham Biosciences).

Subcellular fractionation
Subcellular fractionation (Fig. 4) was performed by stepwise extraction with buffers containing various concentrations of NaCl. Briefly, cells were washed twice with ice-cold PBS and extracted at a density of 2x106 cells/ml with buffer C, previously described as cytoskeletal buffer (29), containing 0.1, 0.2, 0.3, 0.4 or 2 M NaCl with PIC. The pellet was recovered by centrifugation (600 g for 3 min) at 4°C, after each extraction.

Other assays
Telomere seeding and telomere length analysis were performed as described previously (17), except that genomic DNA was restricted with EcoRV and probe Y was used as the probe. Telomerase activity was determined by the modified telomerase repeat amplification protocol assay, as described previously (48). Senescence-associated (SA) ß-galactosidase staining was performed as described previously (20). Protein concentrations were determined by the BCA protein assay (Pierce, Rockford, IL, USA), using bovine serum albumin as the standard.


   ACKNOWLEDGEMENTS
 
We thank Dr Masamichi Oh-Ishi and Dr Hitoshi Iwahashi for helpful suggestions about two-dimensional gel electrophoresis, Dr Tsuyoshi Akagi for providing the retroviral vector, Dr Naohisa Yoshioka for helpful technical advice and discussion, and Dr Yusuke Matsuoka for providing anti-lamin antibody. J.O. is supported by Research Fellowships from the Japan Society for the Promotion of Science. This work was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan and from the Ministry of Health and Welfare of Japan.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +81 298613040; Fax: +81 298612798; Email: mahito-nakanishi{at}aist.go.jp


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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S.-h. Kim, A. R. Davalos, S.-J. Heo, F. Rodier, Y. Zou, C. Beausejour, P. Kaminker, S. M. Yannone, and J. Campisi
Telomere dysfunction and cell survival: roles for distinct TIN2-containing complexes
J. Cell Biol., May 1, 2008; 181(3): 447 - 460.
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