Telomere shortening triggers a p53-dependent cell cycle ... - Nature

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Oncogene (1999) 18, 5148 ± 5158 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc

Telomere shortening triggers a p53-dependent cell cycle arrest via accumulation of G-rich single stranded DNA fragments Gabriele Saretzki1,3, Nicolle Sitte1, Ulrike Merkel1, Reinhard E Wurm2 and Thomas von Zglinicki*,1,3 1

Institute of Pathology, ChariteÂ. Schumannstrasse 20/21. 10098 Berlin, Germany; 2University Clinic of Radiation Therapy, Medical Faculty, ChariteÂ, Humboldt-University, 10098 Berlin, Germany

It has been repeatedly suspected that telomere shortening might be one possible trigger of the p53-dependent cell cycle arrest, although the mechanism of this arrest remained unclear. Telomeres in human cells under mild oxidative stress accumulate single-strand damage faster than interstitial repetitive sequences. In MRC-5 ®broblasts and U87 glioblastoma cells, which both express wild-type p53, oxidative stress-mediated production of single-strand damage in telomeres is concomitant to the accumulation of p53 and p21 and to cell cycle arrest. This response can be modeled by treatment of cells with short single stranded telomeric G-rich DNA fragments. The arrest is transient in U87 cells. Recovery from it is accompanied by up-regulation of telomerase activity and elongation of telomeres. Overexpression of mutated p53 is sucient to reverse the phenotype of inhibition as well as the delayed activation of telomerase. These data suggest that the production of G-rich single stranded fragments during the course of telomere shortening is sucient to trigger a p53 dependent cell cycle arrest. Keywords: telomeres; p53; check point; cell cycle; DNA damage

Introduction Among the many purposes the tumor suppressor p53 serves in a human cell, its involvement in cell cycle arrest following DNA damage is a major one. Which type of DNA damage elicit a p53-mediated cell cycle arrest is not completely clear. Evidence has been presented that one double strand break per cell might be sucient for cell cycle arrest (Huang et al., 1996). On the other hand, many types of DNA lesions are processed via single stranded DNA intermediates, and single stranded DNA seems to be a possible direct signal for cell cycle arrest in Xenopus (Kornbluth et al., 1992), S. cerevisiae (Garvik et al., 1995; Lin and Zakian 1996), and human cells (Huang et al., 1996; Jayaraman and Prives, 1995). Telomere shortening might be another possible mechanism to trigger the p53-dependent cell cycle arrest. Telomeres shorten with each cell division as long as there is no active counteracting mechanism (Harley et al., 1990). There is substantial evidence linking telomere shortening to the loss of proliferative ability which is

*Correspondence: T von Zglinicki 3 These two authors contributed equally to this work Received 3 September 1998; revised 12 April 1999; accepted 12 April 1999

characteristic for senescence of primary human cell strains (Allsopp and Harley, 1995; von Zglinicki et al., 1995; Bodnar et al., 1998). It has been suggested that a critical shortening of telomeres might be recognized as DNA damage by the p53-dependent cell cycle check point system (Shay et al., 1993; Wynford-Thomas et al., 1995; Vaziri and Benchimol, 1996). However, a satisfactory mechanistic model for this recognition has not been found yet. Recent data indicate that telomere shortening proceeds via the production of single stranded intermediates. Terminal overhangs of the Grich strand were demonstrated to exist in both immortal cells and ®broblasts (Makarov et al., 1997). Single stranded damage accumulates preferentially in telomeres of human ®broblasts (Petersen et al., 1998). During DNA replication, telomeric single stranded damage is converted into shortened telomeres (Sitte et al., 1998). It can be assumed that distal single-stranded DNA fragments are liberated during telomere replication. These data raise the question whether single stranded telomeric DNA fragments might be another possible trigger of the p53-dependent cell cycle arrest. Moreover, if telomere damage does play a role in triggering cell cycle arrest, one should expect the ability of a cell to maintain its telomeres to be important for the duration and/or severity of the arrest. While most somatic human cells cannot counteract telomere shortening, immortal cells, among them at least 85% of all tumor cells, do have this possibility mostly by activation of the telomere-elongating enzyme: telomerase (Counter et al., 1994; Shay and Bacchetti, 1997). To examine the interrelationships between telomere single-strand damage, p53-dependent check point control, and regulation of telomere length and telomerase activity, we compared the ability of oxidative stress to arrest the cell cycle and to interfere with telomere maintenance to that of a treatment with synthetic telomeric and non-telomeric oligonucleotides. In MRC-5 human ®broblasts as well as in U87 glioblastoma cells under mild oxidative stress, there is a preferential accumulation of single-strand damage in telomeres. The treatments increase the abundance of p53 and p21 and block the cell cycle. The same response is triggered by treatment of the cells with Grich but not with C-rich, single stranded oligonucleotides. The cell cycle block is abolished by expression of a dominant negative mutp53. U87 tumor cells which recover from the block show up-regulated telomerase activity and lengthened telomeres. On the other hand, telomerase activity is not in¯uenced per se by an accelerated telomere shortening rate or by the presence of single stranded DNA fragments in mutp53 cells. It appears that the regulation of telomerase activity is not

Telomeres and cell cycle arrest G Saretzki et al

directly linked to telomere length in human tumor cells. Rather, it might be part of a p53-mediated response to DNA damage in telomeres. Results Inhibition of proliferation of U87 cells and telomere maintenance under oxidative stress The glioblastoma cell line U87 harbors wild-type, inducible p53 and p21 (Poulos et al., 1996). Hyperoxia

results in inhibition of proliferation of U87 cells after 11+2 days, while after 26+2 days the cells adapt to this chronic stress and proliferate again, albeit at a lower rate (Figure 1a). The levels of p53 and p21 are up-regulated during the inhibition of proliferation (Figure 1b, days 14 ± 18), but decrease to control levels after adaptation (Figure 2b, days 26 and 38). On the other hand, MDA-MB-231 cells, a breast cancer line harboring mutp53, adapted to hyperoxia without any evident cell cycle block (data not shown). Forty per cent hyperoxia irreversibly blocks proliferation of human ®broblasts which lack active

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Figure 1 Chronic hyperoxia blocks transiently the proliferation of U87 cells and increases p53 and p21 levels. (a) Proliferation rate (in population doublings per day, PD/d) of U87 cells vs time under 40% normobaric hyperoxia (®lled circles) and under normoxia (open triangles). Data are from ®ve independent experiments. (b) Levels of p53 and p21 under chronic hyperoxia are estimated by Western blotting. The duration of hyperoxic treatment is given in days. C denotes normoxic controls

Figure 2 Dierent telomere maintenance in U87 and MDA-MB-231 tumor cells under 40% normobaric hyperoxia. (a) Telomere Southern blot of U87 cells. (b) Telomerase activity (TRAP) of U87 cells. (c) Telomere Southern blot of MDA-MB-231 cells. The time of hyperoxic treatment is given in days. C denotes normoxic controls, N denotes negative controls. The positions of a lHindIII marker (23.1, 9.4, 6.6, 4.4, 2.3, 2.0 kb) are indicated. One typical experiment out of three is shown

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Figure 3 Single stranded damage accumulates in telomeres during inhibition of proliferation. (a) DNA from MRC-5 ®broblasts grown under control conditions (C) or under 40% hyperoxia for 28 days (28d) was treated with S1 nuclease in concentrations between 0 and 8 units/plug, Southern blotted, probed with (TTAGGG)4 (upper row) and re-probed with (CAC)6 (lower row). The highest discernible CAC minisatellite band at about 40 kb is indicated by an arrow. The positions of a lHindIII marker are indicated. (b) DNA from U87 cells grown for the indicated times under 40% hyperoxia was treated with 8 units S1 per plug or mock-treated, Southern blotted and probed with (TTAGGG)4 (upper row) and re-probed with (CAC)6 (lower row). (c) Quantitative estimation of the shortening DTRF (in nucleotides) of telomeres after treatment with S1 nuclease. Proliferation of U87 cells was blocked by either 40% hyperoxia (OXY) or by 80 mM H2O2. Blocked samples were taken between days 10 and 22 of hyperoxia (n=16), and at 6 h after H2O2 treatment (n=4). Proliferating samples were taken from untreated controls (CONT, n=20), or from cells adapted to hyperoxia (between days 26 and 60 of treatment n=12), and at day 21 after H2O2 treatment (n=4). Data are mean+s.e.m. A signi®cant dierence to zero (P55%) is denoted by an asterisk


telomerase within 1 ± 3 population doublings. It also leads to a signi®cant acceleration of telomere shortening. Accordingly, telomeres in hyperoxic-blocked ®broblasts are as short as the ones in cells which reach the Hay¯ick limit under normoxic culture conditions (von Zglinicki et al., 1995). In order to see whether activation of telomerase in tumor cells would be able to counteract accelerated telomere shortening, we measured telomere length and telomerase activity in U87 and MDA-MB-231 cells under hyperoxia (Figure 2). In both cell lines, the average telomere length is constant for at least 3 months of culture under normoxic control conditions (data not shown). In U87 cells, the telomere length remains constant even under hyperoxia (Figure 2a) despite an increased rate of single-strand damage (Figure 3b). Telomerase activity is weak in control U87 cells under normoxia and might even slightly decrease during the inhibition of proliferation (Figure 2b, 22 and 27 days). However, telomerase activity is increased signi®cantly in U87 cells which resumed proliferation under hyperoxia (Figure 2b, day 45). In MDA-MB-231 cells, the average telomere length decreased linearly with time at a rate of 17 bp/day (correlation coecient r2=0.917) between about 1 and 3 months of 40% hyperoxia, leading to a signi®cant decrease of the average telomere length in chronically treated cells (Figure 2c). During this whole period, the telomerase activity in MDA-MB-231 cells under hyperoxia remained constant and was not significantly dierent from the activity under normoxia (data not shown). Accelerated telomere shortening under hyperoxia is evidently compensated for by activation of telomerase in U87 cells, but not in MDA-MB-231 cells. Oxidative stress induces single stranded regions in telomeres Single stranded regions accumulate speci®cally in telomeres of human ®broblast (von Zglinicki et al., a

1995, Petersen et al., 1998). In contrast to the bulk of the genome, telomeric single-strand damage is less repaired. It accumulates in cells which do not proliferate and results in an increased rate of telomere shortening in proliferating ®broblasts (Sitte et al., 1998). In order to test whether telomere-speci®c single strand damage correlates with the cell cycle block in telomerase-competent tumor cells as well, we measured the shortening of telomeric fragments after treatment with S1 nuclease. This treatment removes single stranded overhangs (Makarov et al., 1997) and converts interstitial single stranded regions into double strand breaks. The S1 sensitivity of telomeres or CAC minisatellites is hardly discernible in ®broblasts growing under control conditions (Figure 3a). How-

Figure 4 Nuclear uptake of labeled oligonucleotides. MRC-5 cells were treated for 1 h with 10 mM (TTAGGG)2 end-labeled with FITC, ®xed and observed by epi¯uorescence

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Figure 5 Treatment with G-rich oligonucleotides inhibits the growth of MRC-5 ®broblasts (a) and U87 glioblastoma cells (b). Cells were treated with oligonucleotides at the concentrations as indicated. Treatment was done on 4 consecutive days. Proliferation was assayed 1 week after the last treatment as percentage of colonies of more than 50 cells in treated vs untreated cultures. Data are mean+s.e.m. from three independent experiments

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Figure 6 Treatment with (TTAGGG)2 blocks proliferation of MRC-5 and U87 cells. (a) U87 cells (upper row) and MRC-5 ®broblasts (lower row) were partially synchronized by con¯uence, and the DNA histograms were measured 12 h after the release (left column, C12h). At the same time, cells were treated with a single dose of 50 mM (TTAGGG)2. While DNA histograms of untreated controls at 24 h after the release show an increased fraction of S phase cells (middle column, C24h), this increase is largely blocked by the treatment (right column, ODN24h). BrdU pulse labeling (b and c) and cell counting (d and e) show inhibition of growth in treated cells, which lasts for at least 10 days in MRC-5 ®broblasts (b and d), but is overcome after 5 ± 7 days in glioblastoma cells (c and e)


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Figure 7 Involvement of p53 in the oligonucleotide-mediated cell cycle block. (a) U87 cells were partially synchronized and treated with a single dose of 50 mM (TTAGGG)2. Levels of p53 and p21 were estimated by Western blotting at the time after the treatment as indicated (in h). C denotes untreated controls. (b) MRC-5 human ®broblasts harboring wtp53 (®lled circles) and the SV40immortalized subclones MRC-5-SV1 TG1 (open squares) and MRC-5-SV2 (open triangles) were treated with the G-rich oligonucleotide (TTAGGG)2 at the indicated concentrations. Treatment was done on 4 consecutive days. Proliferation was assayed 1 week after the last treatment as percentage of colonies in treated vs untreated cultures. Data are mean+s.e.m. from three independent experiments. (c) Dierent tumor cells lines as indicated in the ®gure were treated with the oligonucleotide (TTAGGG)2. Treatment and assay were performed as above. Open symbols denote mutp53 cell lines, and ®lled symbols denote the U87 cell line harboring wtp53

ever, proliferation of ®broblasts is inhibited after 3 ± 4 weeks of 40% hyperoxia (von Zglinicki et al., 1995), and the telomeres, but not CAC minisatellites, become sensitive to S1 nuclease (Figure 3a). Similar results are found in U87 tumor cells. Telomeres from cells which were proliferation-inhibited by hyperoxia (day 19 of treatment) are visibly shortened by treatment with S1 nuclease (Figure 3b), indicating the accumulation of single-strand damage in telomeres during that period. Minisatellites are at no time sensitive to S1 nuclease (Figure 3b, bottom). The length dierence DTRF between S1-treated and untreated telomeres as measured following oxidative treatments of U87 cells (Figure 3c) con®rms that oxidative stress induces single-strand damage in telomeres of U87 cells. DTRF is found at control level again after release of the cells from the proliferation block, suggesting that the damaged telomeric DNA is lost during replication.

Treatment with short single stranded G-rich DNA fragments inhibits cell proliferation It is known that single stranded DNA fragments can trigger a p53-dependent cell cycle arrest in mammalian cells (Huang et al., 1996). To mimic the production of single stranded DNA fragments under oxidative stress, dierent cell lines were treated with short phosphodiester deoxyoligonucleotides. Using FITC-labeled 12-mer oligonucleotide, nuclear uptake is observed in MRC-5 ®broblasts after 1 h of treatment (Figure 4). C-rich and G-rich telomeric oligonucleotides accumulate equally. The uptake of longer oligonucleotides (24-mers) is considerably less ecient. Similar results were seen using U87 cells (data not shown). Unsynchronized MRC-5 ®broblasts and U87 glioblastoma cells were treated daily for 4 consecutive days with dierent 12-mer oligonucleotides. Treatment with


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Figure 8 Overexpression of mutp53 abrogates the oligonucleotide-induced cell cycle block in U87 cells. U87 parental cells, vector-transfected clones (UV) and mutp53 transfected clones (UP) were partially synchronized by con¯uence and treated with either 50 mM (TTAGGG)2 in late G1 (®lled bars) or with 6 Gy g-irradiation in early G1 (open bars). Proliferation was assessed as the S/G1 ratio in treated vs control cells at 10 and 24 h after the treatments. Data are mean+s.e.m. from both time points and two (irradiation), respective three (oligonucleotide treatment) independent experiments. The expression level of mutp53 is given for each clone in ng/ml lysate with the error of these estimations being about 0.1 ng/ml. Asterisks denote a signi®cant inhibition (P55%)

the G-rich telomeric oligonucleotide (TTAGGG)2 led to a signi®cant proliferation arrest in both cell lines (Figure 5). A G-rich oligonucleotide with the randomized sequence (TGTGAG)2 was slightly less eective than the telomeric sequence. Treatment with the C-rich telomeric oligonucleotide (CCCTAA)2, or with the C-rich oligonucleotide (CAC)4 unrelated to telomeres, did not retard the proliferation (Figure 5). Treatment of U87 cells with phosphorothioate-modi®ed oligonucleotides in concentrations up to 5 mM did not result in a proliferation block. If MRC-5 or U87 cells were partially synchronized by con¯uence at 12 h before treatment, a single bolus dose of 50 mM (TTAGGG)2 was about as eective in blocking proliferation as four consecutive treatments with 30 mM each. This treatment reduces signi®cantly the fraction of cells entering S phase with in the next 12 h (Figure 6a). Moreover, the fraction of cells which incorporate BrdU, indicating active replication of DNA, remains low for a number of days after a single pulse of (TTAGGG)2. Accordingly, cell numbers rise only slowly for at least 6 days after the treatment (Figure 6b ± e). However, U87 glioblastoma cells start to replicate DNA again after day 5 (Figure 6c), and cell numbers increase signi®cantly after day 6 (Figure 6e). On the contrary, the cell cycle block persists over the whole observation period of 11 days in ®broblasts (Figure 6b and d). The oligonucleotide-mediated cell cycle block depends on p53 Both MRC-5 ®broblasts and U87 glioblastoma cells harbor functional active, wtp53 (Asai et al., 1994;

DiLeonardo et al., 1994, and above). Western blots show accumulation of p53 and p21 following treatment of U87 cells with 50 mM (TTAGGG)2 (Figure 7a). Inhibition of growth by treatment with (TTAGGG)2 occurs in MRC-5 parental cells, but not in two SV40 transformed permanent lines derived from it (Figure 7b). Among other eects, SV40T is well known to inhibit the p53-dependent DNA-damage response pathway by binding to both p53 and pRB. Moreover, oligonucleotide treatment did not inhibit colony growth in four dierent tumor cell lines harboring mutp53 (Figure 7c). To examine further the p53-dependency of the oligonucleotide-induced inhibition of proliferation, wtp53 U87 cells were transfected with the mutp53expression vector pC53-SX3 (Baker et al., 1990). Expression of mutp53 was measured in 23 clones by ELISA. In 13 near-diploid clones the amount of mutp53 in the lysate was at or above 500 pg/ml, which is the maximum concentration of wtp53 measured in parental U87 cells after 6 Gy girradiation (data not shown). Six out of these 13 clones were chosen at random and treated with 50 mM (TTAGGG)2 or g-irradiated with 6 Gy (Figure 8). Overexpression of mutp53 abrogates the inhibition of proliferation by either irradiation or treatment with oligonucleotides. Recovery of wtp53 cells from the cell cycle block is accompanied by elongation of telomeres Knowing that telomeric single stranded damage can trigger a cell cycle block via the p53 pathway, the question arises whether up-regulation of telomerase


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Figure 9 Telomere maintenance after transient inhibition of proliferation. (a) Telomere length. Log-phase U87 cells were treated with a bolus dose of 80 mM H2O2 (lanes 1 ± 6). U87 cells at 12 h after release from con¯uence were treated with 50 mM (TTAGGG)2 (lanes 7 ± 9) as were mutp53-expressing U87 clones UP115 and UP132 (lanes 10 ± 13). Telomere length was analysed in untreated controls (C) and at the times indicated (in days) following the treatments. The positions of the l-HindIII size marker (l) are indicated. (b) Telomerase activity of H2O2-treated (lanes 1 ± 5) or (TTAGGG)2-treated (lanes 9 ± 19) U87 cells. (TTAGGG)2treatment was at 12 h after release from con¯uence. Time-matched sham-treated controls (lanes 10, 12, 14, 16 and 18) are shown together with treated cells (lanes 11, 13, 15, 17, and 19) at the indicated times (in days) after the treatment. N, negative control; C, untreated cells; P, positive controls (telomerase activity in 293 kidney tumor cells); R8, concentration standard; TSK1, eciency standard; h denotes heat denaturation of the extract

and re-elongation of telomeres might be part of the release of tumor cells from this inhibition of proliferation. Proliferation of U87 cells was transiently blocked by treatment with either H2O2 or (TTAGGG)2. Cells resumed proliferation after 6 ± 8 days. Telomeres became elongated in U87 cells recovering from a proliferation block induced by treatment with hydrogen peroxide (Figure 9a, lanes 5 and 6) or with G-rich oligonucleotides (Figure 9a, lanes 8 and 9). The mean telomere length increases from 4128+93 bp (n=8) in control U87 cells to 4535+128 bp (n=6, P=0.0215) in those cells recovering from a proliferation block set by treatment with G-rich telomeric oligonucleotides. It increases to 4412+156 bp (n=8) after H2O2 treatment. In contrast, oligonucleotide treatment of the U87

clones UP115 and UP132, which express high amounts of mutp53, does neither inhibit proliferation (Figure 8) nor does it lead to elongation of telomeres (Figure 9a, lanes 10 ± 13). Oligonucleotide treatment of mutp53 MDA-MB-231 cells or SKOV-3 cells, which do not express p53, did neither inhibit proliferation nor did it induce elongation of telomeres (data not shown). These data are corroborated by measurements of telomerase activity: Activation of telomerase accompanies the release from the block following treatments with hydrogen peroxide (Figure 9b, lanes 4 and 5) or with G-rich telomeric oligonucleotides (Figure 9b, lane 19). Telomerase activity is not dependent on the proliferation status of untreated U87 cells after release from con¯uence (Figure 9b, lanes 10, 12, 14, 16, 18). A minor increase of telomerase activity in the treated cells


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was found between 2 and 4 h after treatment with (TTAGGG)2 (Figure 9b, lane 11) which amounted to an average of 2.6+0.8 times of that in controls in ®ve estimates. However, this activation was transient and did not last through the period of inhibited growth (Figure 9b, lanes 15 and 17). Between day 3 and day 7 after the oligonucleotide treatment, telomerase activity in U87 cells as averaged from three independent experiments increases about tenfold (see Figure 9b, lane 19). At day 7, cells had hardly completed more than one population doubling since day 3 (compare Figure 6). Accordingly, induction of telomerase accompanying the release from the block rather than selection of pre-existing cells with high telomerase activity must be responsible for most of the increased activity seen in the TRAP assay. On the other hand, telomerase activity remains constant in clones UP115 and UP132 after treatment with 50 mM (TTAGGG)2 as well as in MDA-MB-231 cells under hyperoxia (data not shown). Discussion The generation of single stranded fragments within telomeres contributes signi®cantly to telomere shortening (Makarov et al., 1997; Sitte et al., 1998; Petersen et al., 1998). The data presented here suggest yet another role: the occurrence of telomeric single stranded DNA, possibly above a certain threshold in length and/or amount, might act as trigger of the p53-dependent cell cycle arrest. Support for this suggestion comes from the following results: (i) Mild oxidative stress triggers the p53-dependent cell cycle arrest even under conditions which produce few, if any, double-strand breaks and few, if any, single strand breaks in the bulk of the genome which are not repaired within a couple of hours. However single stranded regions in telomeres are produced and persist under these conditions. (ii) The p53-dependent cell cycle arrest can be modeled by treatment of cells with G-rich single stranded short DNA fragments. This eect is neither sequence-speci®c, nor completely unspeci®c. It probably requires interstrand G : G base pairing, leading to higher order structures as, for instance, G quartets, which were also proposed for telomeres (Wellinger and Sen, 1997). (iii) Those treatments which produce an abundance of single stranded sites in telomeres or which model the production of these sites not only inhibit proliferation, but, in addition, stimulate telomerase activity in immortal cells during the release from the cell cycle block. This activation is dependent on wtp53. Elongation of telomeres after the treatment of immortal cells with G-rich oligonucleotides was also shown in another study (Wright et al., 1996). However, the p53 status of the cell lines examined was not mentioned, nor was whether or not the cells went through a temporary block of proliferation. Activation of telomerase by DNA-damaging treatments has been found in recent studies (Leteurte et al., 1997; Hyeon Joo et al., 1998). These data suggest a role for telomerase in DNA repair, possibly telomere-speci®c DNA repair, which may make the enzyme important for the resumption of cell growth after a DNA-damaging insult.

The production of single stranded G-rich DNA fragments provides one possible trigger for the p53dependent cell cycle check point system. How this trigger works is not known at present. Although short single stranded DNA can directly bind to the Cterminal domain of p53 (Reed et al., 1995) and can activate its sequence-speci®c double-strand DNA binding (Jayaraman and Prives, 1995), dierent intermediates and co-factors in the signaling pathway from DNA damage to p53 activation are probably necessary. Candidates include the ATM (Mutated in Ataxia Telangiectasia) protein, poly(ADP-ribose)polymerase (Vaziri et al., 1997), and the telomere binding protein TRF2 (van Steensel et al., 1998; Karlseder et al., 1999). Single stranded fragments are produced in telomeres under mild oxidative stress (von Zglinicki et al., 1995; Petersen et al., 1998). Moreover, telomeres accumulate single-strand damage even under standard cell culture conditions (Sitte et al., 1998). Due to their inherent repair de®ciency with respect to single-strand damage, telomeres might act as sensible stress sensors, allowing the cell cycle to pause and repair to take place before serious damage to the bulk of the DNA occurs. Upregulation of telomerase activity and adjustment of telomere length in tumor cells which re-enter the cell cycle is in accordance with such a role for telomeric damage. It is interesting to note that human diploid ®broblasts, in which telomerase cannot be stimulated, are irreversibly blocked by DNA damage induced by either g-irradiation or oxidative stress (DiLeonardo et al., 1994; von Zglinicki et al., 1995; Linke et al., 1997). The resulting arrest phenotype is very similar to replicative senescence (von Zglinicki et al., 1995; Saretzki et al., 1998). Telomeric single-strand damage might be another possible trigger of replicative senescence in addition to chromosome fusion and breakage events following critical shortening of telomeres (Vaziri and Benchimol, 1996; Vaziri et al., 1997). Our data show that telomerase activity in human tumor cells is not necessarily regulated by telomere length per se. Accelerated telomere shortening in the mutp53 line MDA-MB-23 under hyperoxia does not up-regulate telomerase activity, and neither do oxidative damage-modeling treatments in mutp53overexpressing U87 cells. However, telomere maintenance is triggered following activation of the p53 system by telomere damage. This result is in contrast to the situation in yeast (Krauskopf and Blackburn, 1996; Marcand et al., 1997), but in accordance with data showing a p53-dependent telomere length regulation in lung cancer cells (Mukhopadhyay et al., 1998). Materials and methods Cells and culture conditions U87 (glioblastoma astrocytoma), MDA-MB-231 (breast adenocarcinoma), OVCAR-3 and SKOV-3 (ovarian adenocarcinoma), HT29 (colon adenocarcinoma), MRC-5 (fetal lung mortal ®broblasts), MRC-5 SV1 TG1 and MRC-5 SV2 (SV40-transformed MRC-5) were obtained from the ATCC. Both U87 (Asai et al., 1994) and MRC-5 cells harbor wtp53, although the tumor suppressor is blocked by SV40T in


MRC-5 SV1 TG1 and in MRC-5 SV2. In contrast, p53 is mutated in MDA-MDB-231 (Negrini et al., 1994), HT29 (Nagasawa et al., 1995), and OVCAR-3 (Yaginuma and Westphal, 1992), and is not expressed at all in SKOV-3 cells (Yaginuma and Westphal, 1992). Wtp53 U87 cells were transfected by electroporation with the vector pC53-SCX3 which expresses a p53 cDNA pointmutated at codon 143 (resulting in alanine-valine substitution) under the control of the CMV promoter/enhancer (Baker et al., 1990). The vector was gratefully obtained from Dr Bert Vogelstein. Transfections with pcDNA3 (Invitrogen) were used as controls. Fibroblast strains were maintained in Dulbecco's Modi®ed Earle's Medium supplemented with 10% fetal calf serum (Biochrom, Germany), 1% glutamine, and 1% penicillin/ streptomycin. MDA-MB-231, HT29, OVCAR-3 and SKOV-3 cells were maintained in RPMI 1640 containing the same supplements. U87 cells were maintained in Eagle's Minimal Essential Medium supplemented with 5% Basal Medium Supplement, 10% fetal calf serum (Biochrom, Germany), 1% glutamine, and 1% penicillin/streptomycin. Transfected cells were selected using G-418 sulfate (Calbiochem) in a concentration of 500 mg/ml. Chemicals and treatments Chronic hyperoxia was exerted in a three-gas incubator (Nuaire) under a normobaric oxygen partial pressure of 40%. g-irradiation was done at room temperature with a Co60 source at 2 Gy per min. Treatment with hydrogen peroxide (Sigma) was performed in serum-free medium for 30 min. Unsynchronized cells were treated daily with oligonucleotides (BioTez Berlin, Germany) in concentrations as indicated for up to 6 consecutive days. The half-life of these oligonucleotides in serum-supplemented medium has been estimated to be 4 ± 6 h (Wright et al., 1996). For some experiments cells were partially synchronized by con¯uence. They were treated at 12 h after replating with a single bolus dose of 50 mM. To control the cellular uptake, cells grown on coverslides were treated with 10 mM 5'-FITC-labeled oligonucleotides and ®xed after 1 h in ice-cold 70% ethanol. Images were taken in a Zeiss Axiophot microscope (Zeiss Oberkochen, Germany) equipped with a CH250 CCD camera (Photometrics) and a Macintosh Quadra 950 computer. Cell proliferation For DNA ¯ow cytometry, cells were ®xed at the appropriate times in ice-cold 70% ethanol and stained with DAPI. Blue ¯uorescence from at least 104 cells was measured (PAS, Partec GmbH, MuÈnster, Germany). S and G1 phase fractions were calculated using the curve-®tting program provided. Incorporation of bromo-deoxyuridine (BrdU) was measured using a cell proliferation kit (Amersham), and counting at least 2000 cells. Cell growth in mass cultures was assessed by counting at each passage in duplicate using a hemocytometer. Colony counting was used to measure growth inhibition by treatment with H2O2 or with oligonucleotides. Cells were plated at a density of 1 ± 26102 plt cm2 in triplicate or quadruplicate and allowed to settle and grow for up to 2 days before treatment. Colonies were counted 1 ± 2 weeks after the treatments,

depending on the growth rate in the controls. The minimum size for a colony to be counted was 50 cells. Results are expressed as percentage of colonies in the treated vs untreated samples. p53 levels Concentrations of mutp53 in transfected U87 clones were measured by immunoassay (p53 Mutant Selective Quantitative ELISA and Pantropic p53 Quantitative ELISA Assay, Calbiochem). Induction of p53 and p21 by oxidative stress and by treatment with oligonucleotides was controlled by Western blotting. The protein content of cellular lysates was measured by Bradford assay (BioRad) and aliquots corresponding to 26105 cells were subjected to SDS ± PAGE (Laemmli, 1970). Blots were probed with WAF-1 (Ab 1) and p53 (DO1) antibodies (Oncogene Science) and the signals were detected by chemoluminescence. Telomere length, telomere single-strand damage and telomerase activity The mean telomeric restriction fragment length (TRF) and the amount of telomere-speci®c single strand damage were measured as described (von Zglinicki et al., 1995; Sitte et al., 1998; Petersen et al., 1998). Brie¯y, cells were embedded in agarose plugs at a concentration of 56105 cells per plug and digested with proteinase K. The DNA in the plugs was restricted to completion with HinfI and subjected to pulsed ®eld gel electrophoresis under conditions optimized to resolve a size range of 2 ± 30 kb. Gels were blotted and blots were hybridized with a (TTAGGG)4 probe conjugated to alkaline phosphatase. A chemoluminescence signal was generated and recorded on ®lm within the linear density response range. Films were scanned and the mean telomere length was estimated as the weighted average of the optical density as described (Harley et al., 1990). For the measurement of single-strand damage the DNA was subjected to an additional digestion with graded concentrations of S1 nuclease (Petersen et al., 1998). The dierence DTRF between the mean telomere length in untreated controls and the length after treatment with S1 nuclease at saturation concentration was measured. DTRF serves as an estimate of the frequency and/or the length of single stranded regions in telomeres (Petersen et al., 1998). Using the oligonucleotide (CAC)6 as probe for hybridization, the frequency of single strand breaks in nontranscribed minisatellite regions was estimated. Telomerase activity was measured using a semiquantitative Telomere Repeat Ampli®cation Protocol (TRAP) assay (Oncor). Reaction products were resolved on 12.5% polyacrylamide gels and analysed using a phosphoimager (BioRad). The PCR reaction eciency was controlled using an internal standard (TSK1) with each reaction.

Acknowledgements The work was supported by grants from the Deutsche Forschungsgemeinschaft, from VerUm e.V. and from the Berliner Krebshilfe e.V.

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