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(The FASEB Journal. 2003;17:152-162.)
© 2003 FASEB

Induction of a p95/Nbs1-mediated S phase checkpoint by telomere 3' overhang specific DNA

MARK S. ELLER, GUANG-ZHI LI, REZA FIROOZABADI, NEELU PURI and BARBARA A. GILCHREST1

Department of Dermatology, Boston University School of Medicine, Boston, Massachusetts, USA

1Correspondence: Boston University School of Medicine, Department of Dermatology, 609 Albany Street, J-Bldg., Boston, MA 02118-2394, USA.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Telomere shortening induces a nonproliferative senescent phenotype, believed to reduce cancer risk, and telomeres are involved in a poorly understood manner in responses to DNA damage. Although telomere disruption induces p53 and triggers apoptosis or cell cycle arrest, the features of the disrupted telomere that trigger this response and the precise mechanism involved are poorly understood. Using human cells, we show that DNA oligonucleotides homologous to the telomere 3' overhang sequence specifically induce and activate p53 and activate an S phase checkpoint by modifying the Nijmegen breakage syndrome protein, known to mediate the S phase checkpoint after DNA damage. These responses are mediated, at least in part, by the ATM kinase and are not attributable to disruption of cellular telomeres. Based on these and earlier data, we propose that these oligonucleotides mimic a physiological signal, exposure of the telomere 3' overhang due to opening of the normal telomere loop structure, and hence evoke these protective antiproliferative responses in the absence of DNA damage or telomere disruption. Eller, M. S., Li, G.-Z., Firoozabadi, R., Puri, N., Gilchrest, B. A. Induction of a P95/NBS1-mediated S phase checkpoint by telomere 3' overhang specific DNA.


Key Words: DNA damage responses • senescence • ATM • p53 phosphorylation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MAMMALIAN CELLS have multiple complex responses to DNA damage, as well as a tightly regulated program of replicative senescence, all suggested to be fundamental defenses against cancer (1) . In mammals, cell senescence is associated with critical shortening of telomeres, tandem repeats of the DNA sequence TTAGGG that cap the ends of chromosomes (2) and become shorter with each round of DNA replication. In germline cells and most cancer cells, immortality is associated with maintenance of telomere length by telomerase, an enzyme complex that adds TTAGGG residues to the 3' chromosome ends (3 4 5) . The catalytic subunit of telomerase is generally not expressed in normal somatic cells (2) , and after multiple rounds of cell division critically, shortened telomeres trigger either replicative senescence or death by apoptosis, largely dependent on cell type (6 7 8) , although the detailed mechanism is unknown. The mechanism by which telomeres participate in DNA damage responses has been even less clear.

The 3' end of each telomere consists of a single-stranded G-rich overhang that has been proposed to stabilize a loop structure at the chromosome ends by binding of the overhang within the proximal double-stranded portion of the telomere (9) . Two proteins have been identified as having roles in regulating telomere length and integrity respectively: telomere repeat factors TRF1 and TRF2 (10) , which directly bind telomeric DNA. Recently, the POT1 protein was found to be a telomere-binding protein with affinity specifically for the single-stranded repeats of TTAGGG that comprise the 3' overhang (11) . Disruption of the telomere-TRF structure by ectopic expression of a dominant negative mutant version of TRF2 (TRF2DN) results in the exposure and degradation of the 3' telomere overhang, as well as the induction of p53 and, in certain cell types, apoptosis (12) . Although telomere shortening and disruption lead to p53 induction, the feature of these telomeres recognized as DNA damage is not known. Two reports have suggested that exogenously provided telomeric DNA can induce p53 in a sequence-dependent manner. Milyavsky et al. (13) transfected cells with plasmids containing double-stranded telomere repeats and found that these constructs, but not control plasmids containing nontelomeric DNA, induced transcriptionally active p53. Furthermore, Saretzki et al. (14) found that oxidative stress-mediated accumulation of telomeric single-stranded DNA is accompanied by an induction of p53 and that treatment of cells with oligonucleotides containing repeats of TTAGGG also induces p53 and reduces the clone-forming potential of cells in a p53-dependent manner. Although these studies demonstrate that telomeric DNA can induce p53, whether this mechanism is relevant to normal telomere physiology and how it relates to normal telomere structure is unclear. Recently, Blackburn (15) proposed that telomere function is governed not simply by length but also by an equilibrium between a "capped," or silent, state and an "uncapped" state that triggers cell cycle arrest and other DNA damage responses. However, the exact molecular nature of these two states and regulation of their equilibrium are not yet described.

Recently, the p95/Nbs1 protein, mutated in the autosomal recessive disease Nijmegen breakage syndrome (NBS) (16) , was shown to mediate the S phase arrest in response to ionizing radiation (IR) in fibroblasts (17 18 19) . NBS is characterized by mental retardation, immunological deficiencies, an increased cancer incidence, and, at the cellular level, radio-resistant DNA synthesis (16) . p95/Nbs1 is part of a DNA damage complex also containing the Rad50 and hMre11 proteins (20) . This complex exhibits exonucleolytic, endonucleolytic, and DNA unwinding activities (21 22) and is essential for double-strand break repair (20) . In response to IR, the Rad50/hMre11/p95/Nbs1 complex localizes to discrete nuclear foci, presumably the sites of DNA damage (23) . These nuclear foci do not form in irradiated NBS cells (24) . Furthermore, the formation of these foci, as well as the S phase arrest in response to IR, has been shown to depend on the phosphorylation of p95/Nbs1 at serine residues 278 and 343 by the ATM kinase (17) . In addition to its role in the DNA damage response, the Rad50/hMre11/p95Nbs1 complex colocalizes with TRF2 at the telomere during the S phase (25) and may be involved in telomere replication, maintenance, and/or function (26 27 28) . Indeed, in yeast, evidence suggests that the homologs to Mre11 and Rad50 play a role in telomere maintenance (28) .

We have shown that treatment of normal human cells, several mammalian tumor cell lines, or intact rodent skin with small DNA fragments, particularly thymidine dinucleotide, pTT (previously designated pTpT), and more recently other 5–9 base oligonucleotides, causes photoprotective DNA damage-like responses. These include p53 activation (29 , 30 31 32) , increased melanogenesis (tanning) (29 , 33 34 35) , cell growth arrest (34 35 36) , enhanced DNA repair capacity (29 , 30 , 32) , and transient immunosuppression (37) . Many but not all of these responses are accomplished through induction of p53 and p53-regulated genes (31 , 32 , 34) . Noting that all the active but none of the inactive oligonucleotides studied (33 , 35 , 37) have partial sequence homology to the telomere overhang (repeats of TTAGGG), we hypothesized that exposure of this single-stranded telomeric DNA is a critical first signal leading to p53 induction and other events that signal subsequent cancer prevention responses. Furthermore, we proposed that DNA oligonucleotides partially or totally homologous to this 3' overhang mimic the physiological signal in the absence of telomere disruption and therefore similarly stimulate these DNA damage responses. We therefore examined the ability of several oligonucleotides completely or partially homologous to this sequence to induce genes and cell behaviors indicative of DNA damage. We found that such DNA oligonucleotides, in a manner dependent on dose and degree of homology to the telomere 3' overhang, induce the protein levels of E2F1, p53, and p73 and cause an S phase arrest and apoptosis in lymphocytic cells (38) , reproducing the effects of TRF2DN-mediated telomere disruption (12) .

Here we further explore the mechanism by which telomere overhang homologue oligonucleotides induce DNA damage responses. In normal human fibroblasts as well as transformed human cell lines, we detect the induction of the E2F1 and p53 transcription factors. In addition, in fibroblasts p53 is phosphorylated at serine 15, a modification directly associated with increased p53 transcriptional activity (39 40 41 42) . The phosphorylation of p53 on serine 15 induced by the oligonucleotide is mediated by the ATM kinase. Furthermore, both normal and transformed cells display an S phase arrest that is independent of p53 but dependent on p95/Nbs1 and coincides with its phosphorylation in oligonucleotide-treated cells. By cell cycle analysis of control cells lacking p95/Nbs1, we implicate this protein as normally responsible for rapid progression of cells through the S phase and phosphorylation of p95/Nbs1 as temporarily abrogating this function. Finally, we demonstrate that the oligonucleotides induce these responses in the absence of any detectable telomere degradation. These data suggest that exogenously provided TTAGGG sequences mimic the cell signal normally resulting from telomere shortening and/or disruption and might thus be used therapeutically to induce protective antiproliferative cellular responses in the absence of actual changes in genomic DNA damage.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Oligonucleotides
Three DNA oligonucleotides were designed and purchased from the Midland Certified Reagent Company (Midland, TX): one homologous to the telomere overhang (11mer-1: pGTTAGGGTTAG), one complementary to this sequence (11mer-2: pCTAACCCTAAC), and one unrelated (11mer-3: pGATCGATCGAT). Oligonucleotides were resuspended in H2O to give a 2 mM stock solution. For use in experiments, the stock solution was diluted into culture medium, filter sterilized, and added to culture dishes. In all experiments (up to 7 days), cells were given medium containing oligonucleotide only once and were not refed.

Cell sources and culture
Human neonatal fibroblasts were established and cultured as described previously (29) . Fibroblasts from an NBS patient (GM07166) and an age-matched control (GM03399) were purchased from the National Institute of General Medical Sciences (NIGMS) Human Cell Repository, Coriell Institute for Medical Research, Camden, NJ) and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/15% FBS. Fibroblasts from AT patients (GM00367 and GM00647) and age-matched controls (GM01386 and GM 01875) were also purchased from the NIGMS Human Cell Repository and cultured in DMEM/15% FBS. Transformed fibroblasts from an AT patient (GM05849) and an age-matched control (GM00637) were also purchased from the NIGMS Human Cell Repository and were cultured in Eagle’s minimal essential medium-Earle’s balanced salt solution with 2x concentration of essential and nonessential amino acids and vitamins and supplemented with 2 mM L-glutamine and 10% FBS. Saos-2 cells and IMR90 were purchased from the American Type Culture Collection (ATCC; Manassas, VA). SCC12F cells were the kind gift of Dr. James Rheinwald, Harvard University.

Cell cycle analysis
Cell monolayers were harvested, and the nuclear DNA content profile was determined by FACS analysis as described previously (43) . Briefly, cells were collected by trypsinization, fixed in 35% ethanol/65% DMEM, treated with RNase A and stained with propidium iodide. Cells were analyzed using a Becton-Dickinson and Cell Quest software.

Western blot analysis and antibodies
Western blot analysis was performed as described previously (34) . Antibody Ab-6 (DO-1 clone, Oncogene Research Products, Cambridge, MA) detected p53, and antibody KH95 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) detected E2F1. Phospho-p53 (serine 15) was detected using the #9284 polyclonal antibody from Cell Signaling Technology (Beverly, MA). p95/Nbs1 antibody (#NB-100–143, Novus Biologicals, Littleton, CO) and an actin antibody (I-19, Santa Cruz Biotechnology) were also used. Densitometry was performed using a Computing Densitometry System from Molecular Dynamics.

Modification of p95/Nbs1
Jurkat and SCC12F cells were cultured and treated with the oligonucleotides as described above. After the indicated times, total protein was collected and analyzed by 8% PAGE and Western blot using a polyclonal antibody to p95/Nbs1 (#NB-100–143, Novus Biologicals) or a phospho-p95/Nbs1-specific antibody (#3001 Cell Signaling Technology). Immunoprecipitation and treatment with a serine/threonine phosphatase were performed as described by Lim et al. (17) . Phospho-p95/Nbs1 was also detected using the phospho-specific antibody #9284 from Cell Signaling Technology.

Transient expression of TRF2DN
The Ad TRF2DN expression vector was the kind gift of Dr. Titia deLange (Rockefeller University), and the AdGFP was the kind gift of Dr. Cyrus Vaziri (Boston University School of Medicine). IMR90 cells were plated at a density of 3.5 x 103 cells/cm2. One day later, the cultures were given 7 x 1010 viral particles/ml of either AdTRF2DN or AdGFP. Cells were collected at the indicated times after infection, and p95/Nbs1 and p53 were examined by Western blot and cell cycle studies by FACS analysis, as described.

Telomere length
Normal human dermal fibroblasts (passage level 4, ~10 population doublings) were cultured for 5 days in the presence of either diluent or 40 µM 11mer-1, -2, or -3. As a positive control, dermal fibroblasts were cultured for >50 population doublings until senescent (no further increase in cell number after 2 wk in standard medium). The cells were then collected and the genomic DNA isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA). Telomere length was determined essentially as described by van Steensel and de Lange (44) using the Telo TAGGG Telomere Length Assay from Roche Molecular Biochemicals (Indianapolis, IN) and the protocol supplied by the manufacturer. The mean telomere length (MTL) was calculated by densitometry using the method of Harley et al. (45) .

3' overhang assay
Normal human fibroblasts were cultured in the presence of 40 µM 11mer-1 for up to 7 days. Cells were collected before treatment (time 0) and at 3, 5, and 7 days of treatment. The genomic DNA was isolated using the DNeasy kit. Detection of the 3' overhang was carried out as described by van Steensel et al. (10) . A probe ([TTAGGG]4) was hybridized to the genomic DNA to control for hybridization to telomeric double-stranded DNA. The test primer ([CCCTAA]4) was used to detect the overhang.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Karlseder et al. (12) found that human fibroblasts did not undergo apoptosis in response to telomere disruption by TRF2DN nor was there any effect on the cell cycle 72 h after infection with the adenovirus vector expressing this mutant protein. Cell cycle analysis was not presented for earlier time points. However, they did detect an induction of p53 in fibroblasts 24 h after infection. We found that normal neonatal human fibroblasts respond within 24 h to the telomere overhang homologue oligonucleotide 11mer-1 by activation of an S phase checkpoint. This results in an accumulation of cells in the S phase of the cell cycle, reflecting either cessation of DNA synthesis or a slowed progression of cells through the S phase (Fig. 1 A), but no apoptotic cells were detected up to 72 h after treatment. Control 11-base oligonucleotides complementary (11mer-2) or unrelated (11mer-3) to the telomere overhang had no effect on the cells. The selective effect of the telomere homologue oligonucleotide cannot be attributed to selective uptake, as both we (35 , 38) and others (14) have shown comparable uptake among multiple same length oligomers regardless of sequence. This uptake varies somewhat among cell types but is generally rapid with pronounced nuclear localization after 1–4 h (14 , 35) . Also, Wright et al. (46) found no correlation between oligonucleotide stability and ability to induce telomere extension: the half-lives of two 12-mer oligonucleotides, one effective and one ineffective in medium with 10% serum were both 4–6 h. Although this half-life could be extended to 24 h by heat-inactivating the serum used in the culture medium, this decreased the effectiveness of the active oligonucleotide.



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  		Figure 1. Induction of S phase arrest, E2F1 and p53 protein levels, and phosphorylation of p53 in normal human fibroblasts. Preconfluent cultures of normal neonatal human fibroblasts were treated with the oligonucleotides (40 µM) or diluent alone and collected 24 h later for FACS analysis (A) or Western blot (B-D). A) Averages and SD were determined from triplicate samples. B) 1 representative experiment of 3. E2F1 and p53 induction and activation of p53 as indicated by phosphorylation on serine-15 was determined by Western blot analysis, using E2F1, p53, and phospho-p53 (ser-15) specific antibodies. For p53 detection, the blot was first probed for phospho-p53 (serine 15) and then the blot was then stripped and reprobed with the pantropic p53 antibody DO-1. p53-ser 15* = phosphorylated p53. Lanes 1, 2, and 3 contain protein from fibroblasts treated with diluent alone, 11mer-1, or 11mer-2, respectively, collected at the indicated times. As a positive control, fibroblasts from another donor were either sham or X-irradiated (10 Gy) and collected after 3 h. Densitometric analysis of B showed a 170–250% increase in E2F1 in 11mer-1 treated fibroblasts compared to diluent or 11mer-2 treated controls at 24 h p53 increased by 60–300% at 48 h and 240–380% at 72 h after 11mer-1 treatment compared to diluent or 11mer-2 treated controls. All values were normalized to ß-actin and are based on 2 independent experiments. In a separate experiment, cells treated and collected at earlier time points, as indicated, were analyzed for E2F1 (C) and for p53 (D) and p53-serine 15 phosphorylation, as described for B.

Western analysis of the treated fibroblasts showed that p53 and E2F1 are selectively induced by the telomere overhang homologue (Fig. 1B ). An increase in E2F1 is seen at 24 h after addition of the 11mer-1, and not the diluent or control oligonucleotide 11mer-2, and subsequently returns to control levels (Fig. 1B ). Total p53 increased by 48 h and remained elevated for at least 24 h (Fig. 1B ). The E2F1 transcription factor is known to cooperate with p53 in induction of apoptosis (47 , 48) and to induce a senescent phenotype in human fibroblasts in a p53-dependent manner. To determine whether 11mer-1, like pTT (29 , 32) , activates as well as induces p53, we also examined p53 phosphorylation, a marker of transcriptional activation of p53 after various forms of DNA damage (39 40 41 42 , 49) . There was a striking and selective increase in p53 protein phosphorylated at serine 15 in response to the 11mer-1 (Fig. 1B ), which was detected at 24 h and sustained for at least 48 h. To determine more precisely the temporal relationship to addition of the oligonucleotides, these protein inductions and modifications were subsequently examined at earlier times. E2F1 induction (Fig. 1C ) was first detected at 12 h, and p53-serine-15 phosphorylation (Fig. 1D ) could be detected as early as 4 h after addition of the 11mer-1.

Because both p53 (50 , 51) and E2F1 (52) are known to be phosphorylated in an ATM-dependent manner after exposure to IR, we wished to examine the role of ATM in p53 and E2F1 phosphorylation in response to the 11mer-1. Fibroblasts from a patient with Ataxia telangiectasia (AT) known to lack functional ATM protein and normal control fibroblasts were treated with either diluent alone or the 11mer-1, and protein samples were collected for Western analysis. In normal fibroblasts, the 11mer-1 induced a 10–15 fold increase in the level p53 phosphorylation on serine 15 compared with diluent-treated controls (Fig. 2 A). In contrast, treatment of AT fibroblasts with the 11mer-1 resulted in <50% increase. As expected, AT fibroblasts showed only minimal phosphorylation of p53 serine 15 in response to IR. Similarly, E2F1 was induced over 30-fold in an SV40-transformed wild-type human fibroblast line, whereas there was only an ~80% increase in E2F1 in a similarly established AT fibroblast line (Fig. 2B ). Interestingly, in the control wild-type (WT) cells, IR, used as a positive control, resulted in a decrease in E2F1 levels after 3 h, whereas our data with normal fibroblasts (Fig. 1B ) and that of Lin et al. (52) demonstrate up-regulation of E2F1 in response to IR in nontransformed cells. These differences in E2F1 induction in response to IR may reflect disruption of the pRb/E2F1 regulatory pathway due to SV40 T-antigen (53 , 54) . Regardless, these data demonstrate a strong role for the ATM kinase in this response to telomere overhang DNA and are consistent with the previously reported ATM-dependent p53 induction by TRF2DN (12) .



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  		Figure 2. ATM mediates E2F1 induction and p53 phosphorylation by telomere overhang oligonucleotide. To examine the role of ATM in oligonucleotide induction of p53-serine-15 phosphorylation, wild-type (GM01386) and AT (GM00367) fibroblasts were treated with either diluent (lane 1), 11mer-1 (lane 2), 10Gy IR (lane 3), or sham irradiation (lane 4) and were collected after 48 h (oligo-treated) or 3 h (IR) and were analyzed for phosphorylated p53 and total p53 (A). SV40-transformed wild-type and AT fibroblasts (GM00637 and GM05849, respectively) were treated with either diluent (lane 1) or 11mer-1 (lane 2) or were exposed to 10 Gy IR (+) or were sham irradiated (-), collected after 48 h (oligos) or 3 h (IR), and analyzed by Western blot for E2F1 expression (B). Densitometric value of the E2F1 band in each lane was normalized to the value of the ß-actin; normalized values of lanes 1 and 2 were compared with lane 1, whereas IR "+" was compared to IR "-" Ratios, indicating multiples of increase, are shown in B.

To determine whether these oligonucleotides similarly affect other human cell types and to investigate the role of p53, we treated a squamous cell carcinoma line (SCC12F) (55) that overexpresses a presumptively mutated p53 (56) and a p53-null osteosarcoma cell line (Saos-2) (57) with either diluent alone, 11mer-1, 11mer-2, or 11mer-3. Cultures were analyzed by FACS. As with the Jurkat cells and normal fibroblasts, the telomere overhang homologue selectively induced an S phase arrest in both of these cell types within 48 h (Fig. 3 ). The arrest persisted for at least 72 h in Jurkat cells (38) as well as in fibroblasts and Saos-2 cells (data not shown). Interestingly, although the percentage of cells in the S phase in 11mer-1-treated SCC12F and Saos-2 cells was virtually identical (53±6 and 52±1%, respectively), the G0/G1 content and G2/M content of the two cell types were somewhat different. This may reflect differential uptake of the oligonucleotide (35 ; unpublished data), leading to dose-dependent differences, and/or different pathways acting in these cells to control the cell cycle. These data demonstrate that the 11mer-1 affects cells of both epithelial and mesenchymal origin and that the S phase arrest in response to 11mer-1 is not dependent on p53. However, because many cell types with wild-type p53 are relatively resistant to apoptosis after DNA damage (58) , the data do not address the role of p53 in the apoptotic response to 11mer-1, which notably occurs in Jurkat cells (38) that express a presumptively compromised p53 (59) .



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  		Figure 3. Cell cycle arrest is not dependent on p53. A squamous cell carcinoma line (SCC12F, top) with presumptively mutated p53 and a p53-null osteosarcoma cell line (Saos-2, bottom) were treated with either diluent or 40 µM of the indicated oligonucleotide and were collected after 48 h for FACS analysis. For both cell types, percentage and SD of cells in each phase of the cell cycle were calculated from triplicate cultures of each condition. FACS profiles from 1 representative experiment of 3 are shown.

Because of the demonstrated role of p95/Nbs1 in the S phase arrest in response to IR and the known association of this protein with telomeres, we next examined its role in the S phase arrest seen in response to the telomere overhang homologue. Fibroblasts from an NBS patient, in which p95/Nbs1 was below the level of detection (Fig. 4 A), and control normal fibroblasts were treated with the oligonucleotides, and the cell cycle response was determined by FACS. As expected, 11mer-1-treated normal cells exhibit an S phase growth arrest within 24 h, whereas the same cells treated with diluent alone, 11mer-2, or 11mer-3 are unaffected (Fig. 4B ). Interestingly, however, untreated (data not shown) and diluent-treated NBS cells display a FACS profile similar to that of the normal cells arrested in the S phase, and, compared with the diluent-treated NBS cells, their proliferation is minimally affected by the 11mer-1 (Fig. 4B ). These data suggest that p95/Nbs1 has a previously unidentified role in normal progression of the S phase and that DNA synthesis is protracted in the absence of functional p95/Nbs1. Also of interest, the 8% increase in the number of S phase cells in 11mer-1-treated NBS cultures compared to diluent-treated cells, although not statistically significant (P<0.08), suggests that factors other than p95/Nbs1 may contribute to the arrest following DNA damage or 11mer-1 treatment. For example, unscheduled E2F1 activity during the S phase has been shown to lead to activation of an S phase checkpoint (60) . Whether this mechanism is responsible for the modest additional S phase accumulation of NBS cells in response to the 11mer-1 (Fig. 4B ) is now under investigation.



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  		Figure 4. Cells lacking functional p95/Nbs1 have an abnormal FACS profile and respond minimally to telomere overhang homologue. Preconfluent untreated cultures were collected for Western blot analysis using an antibody recognizing total p95/Nbs1 and an actin antibody as a loading control (A). Preconfluent cultures of normal (control) fibroblasts and NBS fibroblasts were treated with 40 µM of the oligonucleotides and collected after 48 h for FACS analysis (B). Averages and SD were calculated from triplicate cultures of each condition. FACS profiles from 1 representative experiment of 3 are shown.

Phosphorylation of p95/Nbs1 by ATM, causally related to activation of the S phase checkpoint after DNA damage from IR, can be detected by PAGE as a subtle slowing of the protein migration in the gel (17 18 19) . Western blot analysis detects such a shift in p95/Nbs1 migration in protein harvested from Jurkat cells 48, 72, and 96 h after addition of the 11mer-1 but not 11mer-2 or 11mer-3 (Fig. 5 A). This change in apparent molecular weight of p95/Nbs1,presumably from covalent modification of the protein, coincides with an S phase arrest as determined by FACS analysis (38) . This shift in p95/Nbs1 migration also occurs in these cells after IR (Fig. 5A ), as previously reported and ascribed to protein phosphorylation (17 18 19) . SCC12F cells respond identically to addition of the 11mer-1 (Fig. 5B ), again in a time frame in agreement with their activation of the S phase checkpoint. The shift in SCC12F cells is more apparent with immunoprecipitated p95/Nbs1 from 11mer-1-treated and irradiated cells (Fig. 5C ) and is eliminated by treatment of the immunoprecipitated protein with a serine/threonine phosphatase (Fig. 5D ), as was reported for p95/Nbs1 phosphorylated in response to IR (17) . Probing a Western blot of proteins from diluent- and oligonucleotide-treated fibroblasts with a phospho-p95/Nbs1-specific antibody further demonstrates that phosphorylation of p95/Nbs1 is specifically induced by the telomere overhang homologue oligonucleotide 11mer-1 in fibroblasts (Fig. 5E and F ) and Saos-2 cells (Fig. 5G ).



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  		Figure 5. Treatment with the telomere overhang homologue leads to phosphorylation of p95/Nbs1. Jurkat (A) and SCC12F (B-D) cells were cultured and treated with the oligonucleotides as described in the legends to Figs. 1 and 3 . At the indicated times, total protein was collected and analyzed by Western blot. Lanes 1, 2, 3, and 4 contain protein from cells treated with diluent or 40 µM of the telomere overhang homologue (11mer-1), complement (11mer-2), or unrelated oligonucleotide (11mer-3), respectively, for the indicated period of time. As a positive control, cells were irradiated with 10 Gy of gamma radiation (+) or were sham irradiated (-) and collected after 3 h. Two forms of p95/Nbs1 are labeled with the asterisk indicating the modified higher molecular weight form. Immunoprecipitation (C) and treatment with a serine/threonine phosphatase (D) were performed with SCC12F cells as described by Lim et al. (17) to confirm loss of the shift when phosphorylation was prevented. Normal fibroblasts (E, F) and Saos-2 cells (G) were cultured and treated with the oligonucleotides. Lanes 1, 2, and 3 contained protein from cells treated with diluent, 11mer-1 or 11mer-2, respectively. Control cells (3 h) were irradiated with 10 Gy of IR (+) or were sham irradiated (-). Phosphorylation of p95–bs1 induced by the telomere overhang oligonucleotide, as well as by IR, was demonstrated by probing the western blots with antibody specific for phospho-p95–bs1 (E-G). Upper band in IR+ and IR-cells in E is not a consistent finding and likely represents a nonspecific interaction with the antibody.

Because p95/Nbs1 is known to be phosphorylated by ATM in response to IR (17 18 19) , we also examined the role of ATM in p95/Nbs1 phophorylation in response to the 11mer-1. Phosphorylation of p95/Nbs1 increased to almost fourfold control levels in wild-type fibroblasts but only by 50% in AT cells (Fig. 6 ). Similarly, phosphorylation of p95/Nbs1 by IR was greatly reduced in the AT cells, as expected. The low level of p95/Nbs1 phosphorylation in these AT cells by the 11mer-1 or IR may reflect low residual levels of ATM activity in these cells or the participation of other kinases in this response. Regardless, these data demonstrate ATM-mediated phosphorylation of p95/Nbs1 in response to treatment with the 11mer-1.



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  		Figure 6. ATM mediates p95/Nbs1 phosphorylation in response to the telomere overhang oligonucleotide. AT and wild-type fibroblasts were treated as described in the legend to Fig. 2B and probed for phospho-p95/Nbs1 and total p95/Nbs1. Phospho-p95/Nbs1 values were normalized to total p95/Nbs1 values and lanes 1 and 2 compared with lane 1 and IR+ compared to IR-. Relative values are shown as ratio to control.

Karlseder et al. (12) previously demonstrated that ectopic expression of TRF2DN, which disrupts the telomere loop structure and exposes the 3' overhang, induces an increase in p53 and apoptosis in certain cell types. To see if p95/Nbs1 and p53 modification are also induced by telomere disruption, IMR90 cells were infected with AdTRF2DN or AdGFP, which expresses green fluorescent protein, as a control. Cells were collected up to 5 days after infection, and p95/Nbs1 and p53 were analyzed by Western blot. By 2 days postinfection, distinct phosphorylation of p95/Nbs1 and p53-serine15 was detected (Fig. 7 A). Phosphorylation of p53-serine 15 was sustained through 4 days postinfection, associated with modest induction of total p53 protein. P95/Nbs1 remained phosphorylated through at least day 5 but with no change in total p95/Nbs1 protein. Similarly, IMR90 cells were infected with AdGFP or AdTRF2DN and were collected for FACS analysis. Concomitant with the phosphorylation of p95/Nbs1, AdTRF2DN infection resulted in a S phase accumulation of cells, most evident on days 4 and 5 postinfection (Fig. 7B ). These data are consistent with those of Karlseder et al. (12) , who found an increase in the percentage of HeLa cells in the S phase 72 h after infection with TRF2DN, the only time point at which cell cycle distribution was reported. Thus, both telomere disruption and treatment with a homologue of the telomere 3' overhang induce modification of p95/Nbs1, leading to partial S phase arrest. These data support our hypothesis that exposure of the 3' overhang is the primary signal for the DNA damage responses observed after various manipulations of the telomere.



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  		Figure 7. Telomere disruption of TRF2DN leads to phosphorylation of p53 and p95/Nbs1 and an S phase arrest. A) IMR90 cells were infected with AdGFP, expressing green fluorescent protein, as a negative control (-) or AdTRF2DN (+). Cells were sham irradiated (IR-) or exposed to 10 Gy (IR+) and collected after 3 h as a positive control. Infected cells were collected up to 5 days, and protein was analyzed by Western blot using antibodies recognizing phospho-p53-serine15 or phospho-p95/Nbs1, as well as antibodies recognizing total p53 or p95/Nbs1. B) IMR90 cells were similarly infected with AdTRF2DN or AdGFP as described above and then collected at the indicated times for FACS analysis. Solid line profiles represent cells infected with AdTRF2DN, and dotted lines represent those receiving AdGFP. Graphs are representative of 2 experiments.

Telomere overhang oligonucleotide does not disrupt telomeres
To eliminate the possibility that the 11mer-1 oligonucleotide acts indirectly by disrupting the telomere loop structure, leading to critical telomere shortening and/or degradation of the 3' overhang as reported after TRF2DN transfection (12) , we analyzed the MTL after treatment of normal human fibroblasts with the oligonucleotides for 5 days, longer than necessary to induce p53 and the S phase checkpoint. None of the oligonucleotides reduce MTL (Fig. 8 A). Indeed, 11mer-1-treated cells showed a modest increase in MTL of ~20% during the 5 day experiment, consistent with previously reported responses of fibroblasts cultivated in the presence of telomere homologue oligonucleotides (14 , 46) . Furthermore, treatment of fibroblasts with the 11mer-1 oligonucleotide for up to 7 days does not result in degradation of the telomere 3' overhang (Fig. 8B ), as is observed following telomere disruption by TRF2DN (12) . These data strongly suggest that the 11mer-1 does not affect telomere integrity but likely mimics the signal created by this process. Similarly, the 11mer-1 effects cannot be attributed to inhibition of telomerase, for example, by acting as a pseudosubstrate and hence preventing telomere elongation, because normal human fibroblasts, cells in which the effects are readily observed, do not express the catalytic subunit TERT (2) . Furthermore, telomere erosion due to telomerase inhibition would be expected to exert effects only after an extended period of time, >50 population doublings in previous reports (61 62 63) , far greater than the 24–48 h reported here, and is in any case not observed (Fig. 8) .



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  		Figure 8. Oligonucleotide treatment does not decrease telomere length or alter the 3' overhang. Normal human fibroblasts were treated with either diluent alone, 40 µM 11mer-1, 11mer-2 or 11mer-3 for 5 days and were then harvested, and the genomic DNA was isolated. A) Telomere length analysis: lane 1, diluent-treated cells; lane 2, 11mer-1-treated cells; lane 3, 11mer-2-treated cells; lane 4, 11mer-3-treated cells; lane 5, late passage fibroblasts (population doubling>50); lane 6, high molecular weight telomere markers; lane 7, low molecular weight telomere markers. MTL (in kilobase pairs) for each experimental condition, calculated from 3 experiments as described by Harley et al. (45) , were as follows: 10.2 ± 0.6 (diluent), 12.6 ± 0.4 (11mer-1), 10.5 ± 0.1 (11mer-2), 10.22 ± 0.01 (11mer-3), 8.86 ± 0.01(senescent control), 10.08 ± 0.01 (high molecular weight standard), and 4.04 ± 0.01 (low molecular weight standard). B) 3' overhang assay was carried out on newborn fibroblasts treated for 3, 5, or 7 days with 40 µM 11mer-1. Lane 1, untreated cells, hybridized with a control overhang-homologue probe; lane 2, untreated cell DNA hybridized with the overhang-detecting, complementary probe; lanes 3–5, DNA from cells treated with the 11mer-1 for 3, 5, or 7 days, respectively, and hybridized with the overhang-detecting probe. Note that lane 5 was digitally transposed in the auto-radiograph to achieve logical comparison sequence with resulting discontinuity of background intensity.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Under normal conditions, the 3' telomere overhang DNA sequence is believed to be folded back and concealed in a loop structure stabilized by TRF2 (9) . However, this TTAGGG tandem repeat sequence might be exposed by critical age-associated telomere shortening that destabilizes the telomere loop structure. This interpretation is consistent with the recent findings of Karlseder et al. (64) that senescence is more closely associated with telomere state than telomere length and that short telomeres can be stabilized by overexpression of TRF2. The repeat sequence might also be exposed if the telomere were distorted, for example, by ultraviolet (UV)-induced thymine dimers or carcinogen adducts involving guanine residues [as with cisplatin or benzo-(a)-pyrene] that could render the loop-back configuration unstable (Fig. 9 ). On the basis of present and previously published data (12 , 29 , 32 33 34 35 36 37 38) , we suggest that exposure of the TTAGGG repeat sequence signals through ATM, p53, E2F1, and p95/Nbs1 to produce a variety of responses, dependent on cell type as well as intensity and/or duration of the signal (Fig. 9) . These responses include cell cycle arrest, apoptosis, possibly senescence, and a more differentiated sometimes adaptive phenotype, for example, increased melanin production (tanning), as we and others have previously shown for melanocytes or intact skin treated with the partial telomere homologs, such as pTT and selected 7mer and 9mer sequences (33 34 35 36) . Whether the ability of telomere homologue oligonucleotides to selectively induce these responses is due primarily to their TTAGGG base sequence or to a tertiary structure resulting from this sequence has not been determined at this time. As in the present experiments, these responses evolve over several days, although in at least some instances specific protein activation and gene induction are observed within 3–6 h (34 , 65) , similar to the time course of cellular responses after DNA damage. As well, we cannot rule out entry into this signaling pathway through the ATM-related kinase ATR known to mediate some responses to UV irradiation (41) , which are mimicked by the telomere homologs.



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  		Figure 9. Proposed mechanism for induction of senescence, cell cycle arrest, adaptive differentiation, or apoptosis by exposure of the single-stranded telomere DNA sequence. The 3' telomere overhang is normally sequestered within a loop structure stabilized by TRF2 (10) , forming a "capped" or silent telomere (15) . Destabilization of this loop structure by DNA damage due to UV irradiation or chemical adducts, expression of TRF2DN (12) , or gradual erosion during aging is hypothesized to expose this single-stranded DNA (repeats of TTAGGG), "uncapping" the telomere. Displacement of the TRF2 protein as shown in the figure might or might not accompany loop disruption under physiological conditions. This single-stranded DNA is then detected by an as yet unidentified sensing mechanism. Interaction of this sensor with the 3' overhang initiates a cascade of events that includes ATM activation, followed by p53 activation, E2F1 induction, and p95/Nbs1 modification, leading to cell cycle arrest. Depending on cell type and/or intensity and duration of the signal, these events might also lead to the eventual induction of senescence, adaptive differentiation, or apoptosis. In the present experiments, we hypothesize that DNA oligonucleotides homologous to the overhang sequence are recognized by the same sensing mechanism, triggering the cascade in the absence of telomere disruption.

It has been reported that exogenously added double-stranded telomeric DNA provided as transfected plasmids can induce an antiproliferative response (13) , but our work and that of Saretski et al. (14) suggest that single-stranded DNA sequences are responsible for this response. Although our data do not rule out additional effects from double-stranded DNA, it is possible that such plasmids require degradation and/or conversion to single-stranded sequences to be active. Alternatively and as proposed by Milyavsky et al. (13) , the double-stranded sequences may compete with telomeres for the telomere-binding factors, disrupting endogenous telomere structure and exposing the 3' overhang, as occurs following transfection with TRF2DN (12) . Interestingly, these investigators ascribe the antiproliferative effect of telomeric DNA to p53. However, their data are largely based on stringently calculated colony-forming efficiencies of treated cells and might not detect transient p53-independent cell cycle arrest. Although FACS analysis performed by Saretzki et al. (14) failed to detect an S phase arrest 24 h after addition of the oligonucleotides, they studied cultures synchronized by confluence, for which the next checkpoint occurs at the G1/S boundary, whereas our cultures were growing exponentially, with cells in all phases of the cell cycle. Conceivably, both p53 and p95/Nbs1 can mediate cell cycle arrest in response to telomere homologue oligonucleotides, with the predominant checkpoint determined by the position of cells in the cycle at the time of stimulation. As well, it must be noted that cells arrested in early S phase are very difficult to distinguish from cells in G0/G1 by FACS analysis. Finally, although not discussed by Milyavsky et al. (13) , their tabular data actually support an S phase arrest in p53 deficient cells treated with telomere homologue DNA.

Experimental telomere disruption (64) and other cellular manipulations that can precipitate premature senescence, such as exposure to oxidative stress (66) , are known to reduce the 3' telomere overhang and/or to shorten overall telomere length. In contrast, exposure of cells to telomere homologue oligonucleotides in the present or previous (14 , 46) studies increases MTL. We interpret the data to suggest that the oligonucleotides activate telomerase, presumably by inducing TERT (67) , and to imply that transient activation of telomerase is part of the physiological telomere-based DNA damage response that also includes activation of the ATM kinase with subsequent signaling through p53 and p95/Nbs1. The apparent ability of oligonucleotides to induce this response in the absence of DNA damage and telomere disruption offers the possibility of "rejuvenating" cells through telomere elongation, as recently reported in human skin equivalent constructs containing fibroblasts transfected with TERT (68) but without the enhanced risk of carcinogenesis observed in even normal cells that ectopically and continuously express telomerase (69 , 70) . Equally, the phenomenon suggests that the advantages of robust DNA damage responses of the type observed in p53 overexpressing mice can be separated from the premature senescence also observed in these transgenic animals (71) .

Our model predicts that inability to repair damage to telomeric DNA would lead to exaggerated damage responses, such as p53 induction and apoptosis, as has been reported for UV-irradiated xeroderma pigmentosum cells that cannot efficiently remove DNA photoproducts (49) . Further, it is tempting to speculate that a DNA damage recognition mechanism might evolve to contain predominantly thymidine and guanine bases. The TTAGGG sequence is an excellent target for DNA damage, as dithymidine sites most commonly participate in formation of UV photoproducts (72) and guanine is both the principal site of oxidative damage, forming 8-oxoguanine (73) , as well as the base to which most carcinogens form adducts (74) .

Of interest, the postulated telomere-initiated signal transduction pathway would provide a single evolutionary point of departure for several distinct defenses against carcinogenesis in higher organisms: permanent loss of proliferative capacity (senescence) in cells expected on a statistical basis alone to have accumulated multiple mutations throughout the genome during prolonged environmental exposure and serial rounds of DNA replication and cell division (1) ; transient cell cycle arrest to increase the time available for repair before resuming DNA replication; activation of a cell suicide program (apoptosis) to remove cells from the tissue if damage exceeds the repair capacity, because acute telomeric damage would be expected to reflect the degree of DNA damage throughout the genome; and induction over several days of adaptive responses such as tanning (56) that reduce DNA photoproduct formation (75 , 76) and/or enhance DNA repair capacity (29 , 30 , 32) to prevent damage from similar insults in the future.


   ACKNOWLEDGMENTS
 
The authors acknowledge the excellent technical assistance of Lee Hughes and Hua Tan. This work was supported in part by grants from the Carl J. Herzog Foundation (to B.A.G.) and the National Cancer Institute (to the Boston University School of Medicine Cancer Center).

Received for publication May 21, 2002. Accepted for publication September 29, 2002.


   REFERENCES
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 DISCUSSION
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