Telomerase Activity During the Cell Cycle and in Gamma-Irradiated Hematopoietic Cells F Leteurtre1,Xli1, E Gluckman2 and ED Carosella1

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Telomerase activity during the cell cycle and in gamma-irradiated hematopoietic cells F Leteurtre1,XLi1, E Gluckman2 and ED Carosella1

1Service de Recherches en He´matoImmunologie, Laboratoire d’Immunoradiobiologie, DRM-DSV-CEA; and 2Service de Greffe de Moe¨lle, Hoˆpital Saint-Louis, Paris, France

Telomeric DNA protects chromosome ends from recombination would be expected in telomerase-positive cells (review in events and its length serves as a mitotic clock that triggers exit Ref. 4), however, data concerning the regulation of telomerase from the cell cycle when telomeres become too short. Telomer- 5,6 ase is the enzyme involved in telomere elongation, one of the activity during the cell cycle are contradictory. events that permits unlimited cell proliferation. Variations in Radiation induces DNA breaks that must be repaired in telomerase activity were quantified in hematopoietic cell lines order for a cell to survive. Double-strand breaks (DSB) are the after gamma-irradiation. Telomerase activity increased after most difficult lesions to repair and are assumed to be the most irradiation of between 0 and 3 Gy in a dose-dependent manner, cytotoxic, since the presence of a single unrepaired DSB is reaching a maximum at 3 Gy. The increase in telomerase sufficient to induce cell death. Genomic DSB repair involves activity was nearly maximum 8 h after irradiation, the peak being observed at around 24 h. Although this kinetics partly either homologous or illegitimate recombinations. Most DSB in telomeric sequences are repaired via these general path- correlated with cell redistribution into the G2/M phase of the cell cycle, telomerase activity did not show significant variation ways. In a yeast strain deficient for recombination pathways, over the cell cycle. Therefore, the activation of telomerase an additional possibility has recently been documented: de observed after gamma-irradiation may suggest the involvement novo telomere formation.7 De novo telomere formation has of telomerase in DNA repair and chromosome healing. also been observed to stabilize broken chromosomes in mam- Keywords: gamma-irradiation; telomerase; cell cycle malian cells.8 Telomerase therefore might act as a repair enzyme in cells that express it. In this work we measured telomerase activity in response Introduction to ␥-irradiation of cells. We chose hematopoietic cell lines for the model, since it has been shown that telomerase activity is Telomeres are specific DNA structures consisting of hundreds present in most hematopoietic cells.9–13 Telomerase activation of repeating hexanucleotides, (TTAGGG)n, spanning several could result from the need of cells to repair a broken chromo- kilobases, that cap chromosome extremities. The two princi- some, whereas telomerase down-regulation could result from pal functions of telomeres are (1) to mark chromosome ends, a reduction in the cell proliferation index. While we found thereby protecting them from nuclease degradation and pro- telomerase activity to be increased following gamma- cessing by cell repair machinery, avoiding the loss of ends irradiation and to correlate with radiation-induced cell cycle and of end-to-end chromosome fusions and rearrangements; redistribution into the G2/M phase, it did not vary during the and (2) to serve as mitotic clocks that monitor the number of cell cycle. These results may therefore suggest a role for telo- cell divisions. Indeed, since DNA polymerases require primers merase in DNA repair. for initiation and synthesize DNA in the 5Ј-to-3Ј direction, telomeric DNA can not be completely duplicated at the 5Ј ends, which leads to progressive telomere shortening. In order to avoid the confusion of a short telomere end with a DNA Materials and methods double-strand break (and its deleterious consequences on gen- etic stability), the presence of a short telomere signals cell Cell culture cycle withdrawal. Thus, unrestrained cell proliferation can only be achieved by telomere elongation, either by recombi- KG1a and CEM cells were cultured in Iscove’s modified nation or, more frequently, by the action of a specialized DNA Eagle’s or RPMI 1640 media supplemented with 20% or 10% polymerase, telomerase.1,2 heat-inactivated fetal bovine serum, respectively. Cells were Telomerase is a ribonucleoprotein with an RNA template incubated at 37°C in a humidified atmosphere containing 5% that directs synthesis of telomeric repeats at chromosome CO . All experiments were carried out using exponential- extremities.3 Its activity is highly controlled, being down-regu- 2 phase growing cells. Cells were regularly checked for con- lated in most human tissues, whereas it is re-expressed in tamination by mycoplasma. many cancer cells, in which it is frequently a marker of cell invasiveness (review in Ref. 4). In vitro, terminal differentiation of most cancer cell lines has been reported to down- regulate telomerase activity (review in Ref. 4). Therefore, to Irradiation protocol some extent, telomerase activity can be linked to the status of cell proliferation. Since telomere replication takes place in Cells were irradiated at a concentration of 106 cells per milli- late S phase, telomerase activity in this phase of the cell cycle liter in culture medium. Gamma-radiation doses administered varied from 0 (control) to 4 Gy at a rate of 0.8 Gy per min, using an IBL437C irradiator (CIS Bio-international, Gif-sur- Correspondence: F Leteurtre, SRHI/LIRB/DSV/CEA, Institut d’He´matol- 137 ogie, Hoˆ pital Saint-Louis, 1, avenue Claude Vellefaux, 75475 Paris Yvette, France) equipped with a Cs source. The homogen- ce´dex 10, France eity of sample irradiation, measured by dosimetry using lith- Received 29 October 1996; accepted 28 May 1997 ium fluoride, was greater than 95%. Up-regulation of telomerase activity in irradiated hematopoietic cells F Leteurtre et al 1682 Measurement of cell proliferation by thymidine blocked in the G2/M phase were obtained using nocodazole incorporation for 24 h. Cell cycle analyses were carried out using the BrdU incorporation technique, as previously described.16 For each After irradiation, the cells were seeded on 96-well culture kinetic point, one million cells were incubated for 15 min in plates. In order to allow exponential cell proliferation over a the presence of 30 ␮g/ml of BrdU prior to fixation in 70% cold 5-day period, the number of seeded cells varied according to ethanol for at least 18 h at 4°C. Analyses were performed the dose of radiation: 0.25 × 105 for 0 to 1 Gy, 0.5 × 105 for using a rat BrdU antibody obtained from Seralab (Asnie`res, 2 Gy, 105 for 3 Gy, and 2 × 105 for 4 Gy, respectively.14 At France) and propidium iodide. Cell cycle analysis was carried day 5 of culture, 1 ␮Ci of methyl-3H-thymidine (specific out by flow cytometry, using a FACS Vantage device with activity 25 Ci/mmol; Amersham, Les Ulis, France) was added Lysis II and Cellfit software (Becton Dickinson, Erembode- to each well. The cells were harvested after 4 h and loaded gem, Belgium). onto paper filters. The filters were extensively washed with water, dried and transferred to scintillation vials, after which their radioactivity was quantified. These quantifications, nor- malized to the number of seeded cells, reflected cell prolifer- Results ation. Residual cell proliferation capacity after irradiation, a measure of cell survival, was expressed as the ratio of the Cell survival after irradiation irradiated vs non-irradiated cell proliferation multiplied by 100. Since hematopoietic progenitor cells are one of the main tar- In order to measure the rate of DNA synthesis following gets in cancer radiotherapy and since most hematopoietic irradiation, 3H-thymidine labeling indexes were determined cells display telomerase activity, we used hematopoietic cell over the first 24 h. The protocol used was similar to that lines to test the effects of radiation on such activity. We used described above, except that 3 × 105 cells per well were the KG1a human cell line, which displays an immunopheno- seeded and thymidine was added for only 1 h. type similar to that of hematopoietic stem cells,14 and the CEM cell line, a human T lymphoblastoid cell line. Long-term cell survival after irradiation was determined by the measure of Measure of telomerase activity by the TRAP assay methyl-3H-thymidine incorporation 5 days after gamma- irradiation. The mean survival rate for KG1a cells in three sep- Cells studied for telomerase activity were processed as pre- arate experiments was 91.6 (±8.7)%, 87.6 (Ϯ15.0)%, 50.0 viously described by Kim et al.15 Each sample of 106 cells was (±9.8)%, 16.5 (±5.0)%, and 5.2 (±3.9)% for radiation doses of washed once in PBS, treated with 20 ␮l ice-cold lysis buffer 0.5, 1, 2, 3 and 4 Gy, respectively. CEM cells displayed a simi-

(0.5% CHAPS, 10 mM Tris-HCl pH 7.5, 1 mM MgCl2,1mM lar radiation-dose survival curve (97.5, 35.9, 14.8 and 3.2% EGTA, 10% glycerol, 5 mM beta-mercaptoethanol, and 1 mM after 1, 2, 3 and 4 Gy, respectively). PMS) and incubated on ice for 30 min. After centrifugation at Short-term cell survival was also measured by trypan blue 15 000 g for 30 min at 4°C, the supernatant was stored at exclusion. For KG1a, cell mortality was less than 5% in all −80°C. TRAP assays were carried out on 2 ␮l of diluted samples, except after 48 h in irradiated samples at doses of 2, extracts (corresponding to between 10 and 104 cells) using the 3 and 4 Gy, in which membrane integrity was lost in, respect- TRAP-eze telomerase detection kit (Oncor, F67402, Illkirch, ively, 7.6, 12.2 and 11.4% of the cells. For CEM, cell mortality France). TS primer was elongated for 30 min at 30°C. Twenty- was close to 5% in all samples, except after a 4 Gy irradiation five PCR cycles (for KG1a cell extracts) and 26 PCR cycles at 24 h (8.5%) and 48 h (10.7%). Radiation was therefore seen (for CEM cell extracts) were run in the presence of 4 ␮Ci ␣32P- to reduce the proliferation capacity of KG1a and CEM cells dCTP (3000 Ci/mmol). Temperature steps for each cycle were with only minor immediate effects on cell viability. 94°C for 40 s, 50°C for 40 s, and 72°C for 90 s. Electro- phoresis of the PCR products was carried out with 12% non- denaturing acrylamide gels. Signal intensities on autoradio- grams were quantified using a Molecular Imager system Determination of telomerase activity (Biorad, Hercules, CA, USA). Telomerase activity was measured as the ratio of the radioactivity in the ladder Quantification of telomerase activity using the TRAP assay has (corresponding to the telomerase products) and in the internal been reported to be difficult because of the non-linearity of standard band (the bottom band of the gel), corrected for the test with increasing amounts of cells.17 The incorporation background signals. Variations in telomerase activity were of an internal standard, as with the Oncor TRAP-eze telomer- expressed relative to the appropriate controls (mock irradiated ase detection kit, has been reported to improve the linearity.17 samples at the same kinetic point for irradiation experiments, In our experiments, the telomerase signal was strictly pro-

and at T0 for synchronization experiments). portional to the number of cells (for up to 30 cells in the absence of an internal standard and for up to 300 cells in the presence of one (Figure 1)). Above these thresholds, a two- Cell synchronization and cell cycle analysis fold reduction in test sensitivity was observed for a 3.3-fold increase in the number of cells (to 102 and 103 cells, Exponentially growing cells were synchronized using two respectively). Telomerase assays reported in this paper were 16-h thymidine blocks (0.1 mM), separated by a drug-free per- carried out in the presence of an internal standard within the iod of 10 h. Release from the second thymidine block was linearity range of the test. The variability of the TRAP-eze

taken as time zero. CEM cells chemically blocked in the G2/M assay was also checked and found to be less than 15% for the phase were obtained by adding 200 ng/ml of nocodazole same cell extract (n = 3) and 30% for different cell extracts of (Sigma, St Quentin Fallavier, France) at the end of the S phase, the same batch of cells (n = 5). These levels of variability were

8 h after release from a G1/S block. KG1a cells chemically quite high compared to those encountered using biochemical Up-regulation of telomerase activity in irradiated hematopoietic cells F Leteurtre et al 1683 techniques. For this reason, samples were quantified twice KG1a and CEM cells following gamma-radiation. Only a slight and experiments carried out three to six times. Under these reduction in DNA synthesis was observed 8 h after irradiation conditions, it was possible to demonstrate a two-fold increase (labeling indexes between 0.9 and 0.84 of control values in in telomerase activity. CEM cells and between 1.09 and 0.92 of control values in The activity of telomerase in CEM and KG1a cells, as well KG1a cells). However, the reduction in DNA synthesis was as in other immortal cell lines, was found to be at least two greater 24 h after IR (labeling indexes between 0.84 and 0.36 orders of magnitude greater than in resting PBL. The level of of control values in CEM cells and between 0.71 and 0.42 of activity measured in CEM and KG1a cells were of a magnitude control values in KG1a cells). Thus, a reduction of DNA syn- almost the same as that encountered in activated lympho- thesis was observed following irradiation in the KG1a and cytes. CEM cell lines. The nature and kinetics of the delay were then studied by FACS cytometry in KG1a and CEM cell lines in order to determine whether telomerase up-regulation might correlate with Determination of telomerase activity in KG1a and the accumulation of cells in a specific phase of the cell cycle. CEM cells after gamma-irradiation Complete results are reported in Table 1 and the effects of 3 Gy irradiation are emphasized in Figure 4. In the KG1a cell Telomerase activity was measured several times during the line, the presence of a G /M phase delay was not visible at first 48 h after gamma irradiation (Figure 1a). A significant 2 8 h and was maximum at 24 h, at which time 45% of the cells increase in activity was observed in KG1a and CEM cells were in the G /M phase, decreasing to 30% after 48 h. Thus, (Figure 2) (Student’s t-test, P Ͻ 0.05, four to seven separate 2 in KG1a cells, a strong correlation between the number of irradiations at each dose). These up-regulations were found to cells in the G /M phase and telomerase activity was observed be dose-dependent when irradiation was increased incremen- 2 only at 24 and 48 h after irradiation. On the contrary, 8 h after tally from 0 to 3 Gy, and was less marked at 4 Gy. The magni- irradiation, only a few KG1a cells were in the G /M phase, tude of telomerase up-regulation was more than three-fold 2 relative to controls in both cell lines at 24 h (Figure 2). Kinetic whereas telomerase up-regulation had almost reached its studies of telomerase activity revealed that up-regulation was maximum. In the CEM cell line, the existence of a G2/M block detectable after 4 h (data not shown), was nearly maximal at lasting for approximately 24 h was clearly visible. The fraction 8 h, reached a maximum at 24 h, and then slowly of cells in the G2/M phase increased from 8% in the control decreased (Figure 3). cells to 32% 8 h after irradiation, reaching 41% after 24 h. Only 16% of the CEM cells were found to still be in G2/M 48 h after 3 Gy radiation, by which time the block had loos-

ened. Thus, the maximum accumulation of CEM cells in G2/M Redistribution of cells within the cell cycle following was observed earlier than in KG1a cells and the correlation irradiation between telomerase up-regulation and the number of CEM cells in G2/M at 8, 24 and 48 h post-irradiation were excellent. 3 Radiation induces G1 or G2 delays in the cell cycle. First, H- The kinetics of telomerase up-regulation and cell cycle thymidine labeling indexes were determined, in order to accumulation in the G2/M phase of the cell cycle therefore appreciate the rate of DNA synthesis following irradiation. A were well-correlated in both cell types, except at 8 h in dose-related decrease in DNA synthesis was observed in KG1a cells.

Table 1 Effects of gamma-radiation on cell cycle distribution in KG1a and CEM cells

IR dose 8 h 24 h 48 h

G1 SG2MG1 SG2MG1 SG2M

KG1a 0 Gy 61 34 5 55 39 6 57 34 9 1 Gy 52 40 8 55 36 9 61 18 21 2Gy 553510413029302644 3 Gy 54 40 6 30 25 45 42 28 30 4Gy 493912333334542719 Variation & = ⑀%&&%⑀&⑀&%

CEM 0 Gy 42 50 8 36 56 8 34 60 6 1Gy 34452031571236577 2Gy 245323295219385012 3Gy 125632213841364816 4Gy 145927284725384721 Variation & = %&=%=⑀&⑀%

Measurements were carried out using the BrdU technique, 8, 24 and 48 h after gamma-radiation at doses of between 0 and 4 Gy. Results are expressed as percentages of the cell population. Accumulation of cells in the G2/M phase was measured between 8 and 48 h post- irradiation and was found to be maximal at around 24 h (somewhat earlier in CEM cells than in KG1a cells). Differences at T8 might have been due to the percentage of cells in S phase at the time of irradiation. Doubling times are similar for both cell lines (24 h for CEM, and 26 h for KG1a). Up-regulation of telomerase activity in irradiated hematopoietic cells F Leteurtre et al 1684

Figure 1 (a) Autoradiography showing telomerase activity in KG1a cells using the PCR-based TRAP assay, which includes an internal standard. The telomerase signal is visualized as a ladder; the lowest band corresponds to the internal standard. Left panel: Telomerase activity observed with increasing amounts of cell extract, corresponding to between 10 and 104 cells. Negative controls consisted of heat- inactivated cell extract (C1) and of cell-free extract (C2). The number of PCR cycles was 25. Right panel: Alterations in telomerase activity 24 and 48 h after gamma irradiation at doses of 0 to 4 Gy, as measured in 100 cells. Irradiation increased telomerase activity. (b) Telo- merase activity with increasing amounts of cell extract. Quantiﬁcation of the left side of (a) was carried out. A linear dose–response curve was observed with up to 300 cells in the presence of internal standard (᭹), but only with up to 30 cells in the absence of internal standard (᭺).

Determination of telomerase activity in chemically thymidine block, the cells progressed in an orderly fashion

synchronized cellls from S phase (for the ﬁrst 6 to 7 h) to G2/M (around 8 h), and returned to G1 after 12 h (Figure 5a). Measures of telomerase Since redistribution into the G2/M phase of the cell cycle activity at times 0, 4, 8 and 12 h following release from the mostly correlated with the peak of telomerase activity, we thymidine block did not show signiﬁcant variations examine the enzyme’s activity in various phases of the cell (Figure 5b). Since telomerase activity in cells entering the S cycle. We were unable to chemically synchronize the KG1a phase did not change for at least 12 h, either in the presence cells, therefore, we only measured telomerase activity in or absence or thymidine (data not shown), the cell synchroniz- synchronized CEM cells. Following release from the second ation process had not altered telomerase activity. Thus, telo- Up-regulation of telomerase activity in irradiated hematopoietic cells F Leteurtre et al 1685

Figure 2 Effect of increasing doses of gamma irradiation on telomerase activity in KG1a cells (a) and in CEM cells (b). Twenty-four hours after irradiation, telomerase activity signiﬁcantly increased for all radiation doses (Student’s t-test, P Ͻ 0.05). The increase was dose- dependent at between 0 and 3 Gy (maximum at 3 Gy). Average values of four to seven separate experiments are presented.

Figure 3 Kinetics of telomerase up-regulation following irradiation merase activity was found to be stable during the cell cycle. in KG1a cells (a) and in CEM cells (b). Telomerase activity was measured 8, 24 and 48 h after irradiation. Irradiation doses were 0 (used Telomerase activity was measured in cells that had been as control), 0.5 (᭺), 1 (᭹, dashed line), 2 (ᮀ), 3 (᭿, dashed line), and blocked in G2/M phase by nocodazole in order to determine 4Gy(̄, bold line). Irradiation induced a dose-dependent increase whether a block alone could alter the enzyme’s activity. A 24- in telomerase activity between 0 and 3 Gy that lasted for at least 48 h. h block induced a reduction in telomerase activity to a level Averages and standard deviations obtained from four to seven separ- that was approximately 40% of that in the control cells, 39% ate experiments are presented. of that in the KG1a cells (n = 2), and 41% of that in the CEM cells (n = 2). Telomerase activity was measured in synchron- to a modiﬁcation in the distribution over the cell cycle or to ized CEM cells exiting S phase and encountering the nocoda- a long-lasting block per se. zole block for 8, 16 and 24 h. Only a 24-h block was able to induce a variation in telomerase activity, as mentioned above.

Thus, a long-lasting G2/M block was necessary in order to alter Discussion telomerase activity. The effects of such a block was seen to be a reduction in telomerase activity. The increase in telomerase Telomerase is a highly regulated enzyme. In most human activity observed following irradiation was therefore not due tissues, except in germinal cells and perhaps stem cells,15 telo- Up-regulation of telomerase activity in irradiated hematopoietic cells F Leteurtre et al 1686

Figure 4 Cell cycle redistribution following a 3-Gy irradiation in KG1a (a) and CEM (b) cell lines. The kinetics of cell cycle accumulation

in the G2/M phase of the cell cycle was well correlated with the kinetics of telomerase up-regulation for the two cell lines, except at 8 h post- irradiation in KG1a cells. Up-regulation of telomerase activity in irradiated hematopoietic cells F Leteurtre et al 1687

Figure 5 Telomerase activity in synchronized CEM cells. (a)

Chemical synchronization at the G1/S transition phase was accomplished using a 0.1 mM double thymidine block. Cell distribution in the cell cycle was assessed by FACS analysis immediately after release from the thymidine block (T0), and after 4, 8 and 12 h. Cells were at the G1/S phase transition at T0, into S phase at T4, mostly in the G2/M phase at T8, and had mostly returned into G1 at T12. (b) Autoradiography showing telomerase activity 0, 4, 8, 12, 15 and 24 h after release from a double thymidine block. No significant variation in telomerase activity was observed as cells progressed through the cell cycle. merase activity has been ascribed to down-regulation after birth. Reactivation of telomerase has been detected in most cancerous cells and tissues.9–11,18 Telomerase activity has thus been deemed necessary for unlimited cell proliferation in order to compensate for telomere shortening, which leads to cell cycle withdrawal and cell senescence. In contrast, telomerase down-regulation in somatic tissues was seen as a means for limiting carcinogenesis.19 The hematopoietic system is quite special, since most of its cells express telomerase activity.9–12 More recently, regulation of telomerase activity over time as a function of proliferation status has been reported (review in Ref. 4). Indeed, terminal differentiation of immortal cell lines leads to cell cycle withdrawal and telomerase down-regulation.20,21 Also, activation of T lymphocytes induces cell proliferation and dramatic up-regulation of telomerase activity.11 Finally, data concerning variations in telomerase activity during the cell cycle are contradictory.5,6 In this paper we show that telomerase activity does not change over the cell cycle and that radiation induces up-regulation of telomerase activity in hematopoietic cell lines. In the KG1a and CEM hematopoietic cell lines, which express telomerase activity, gamma-irradiation of between 0 and 3 Gy increased telomerase activity in a dose-dependent manner. This increase was found to be reduced at the highest radiation dose (4 Gy). We did not find that telomerase up- regulation resulted from the onset of cell death by apoptosis, as recently suggested,22 for the following reasons. First, cell viability, as assessed by trypan blue exclusion, was not sig- nificantly changed during the first 24 h post-irradiation, when telomerase activity increase was already maximal. Second, the increase was observed for low radiation doses that have very little effect on cell survival as assessed by cell proliferation capacity 5 days after irradiation. Third, we previously showed Up-regulation of telomerase activity in irradiated hematopoietic cells F Leteurtre et al 1688 that KG1a cell death following radiation did not involve DNA cesses, or to a lesser extent, to be directly involved in the fragmentation during the first 24 h, and only marginally after repair process itself. The telomerase up-regulation we 48 h,14 when telomerase activity was no longer at its observed in response to irradiation might be part of this puta- maximum. tive role for telomerase in the DNA protection/repair process. The post-irradiation increase of telomerase activity was In conclusion, we report an increase in telomerase activity detectable after 4 h, reached a maximum at between 8 and following cell irradiation that was not related to a variation 24 h, and then gradually decreased. Observation of this kin- in telomerase activity consecutive to cell cycle redistribution. etics, which was found to be perfectly correlated to CEM cell These results question the putative role of telomerase in the

accumulation in the G2/M phase following irradiation, DNA protection/repair processes. Additional studies are prompted us to examine telomerase activity in each phase of required before telomerase inhibitors are used in cancer the cell cycle. No variation in telomerase activity was therapy. detected in chemically synchronized CEM cells (in agreement 6 with Holt et al ) or as the result of a G2/M block per se.In addition, in KG1a cells, telomerase up-regulation was Acknowledgements observed 8 h after irradiation, before reduction in DNA synthesis or redistribution of the cell cycle could be detected. We would like to thank Catherine Menier for her contribution Therefore, telomerase up-regulation was not due to the to the FACS analyses, and Ge´rard Socie´ for helpful dis- alteration of cell cycle redistribution upon irradiation. cussions. Financial support was obtained from Electricite´ de Telomerase is up-regulated in response to cell activation11 France and from l’Association pour la Recherche contre le as the result of gene translation.6 Progression past the restric- Cancer. tion point is required for telomerase activation, since rapamy- cin (an inhibitor of p70S6 kinase, a kinase that controls the up- regulation of protein synthesis around the restriction point) References inhibits telomerase activation, but neither aphidicolin nor hydroxyurea (S-phase inhibitors) does.23–25 The presence of 1 Harley CB, Villeponteau B. Telomeres and telomerase in aging resting cells that could enter the cell cycle upon irradiation and cancer. Curr Opin Genet Dev 1995; 5: 249–255. 2 Zakian VA. Telomeres-beginning to understand the end. Science might explain the telomerase up-regulation observed in our 1995; 270: 1601–1607. experiments. However, since exponentially growing cells 3 Feng JL, Funk WD, Wang SS, Weinrich SL, Avilion AA, Chiu CP, were used, no resting cells capable of entering the cell cycle Adams RR, Chang E, Allsopp RC, Yu J, Le S, West M, Harley CB, were present, as verified by the measurement of DNA Andrews WS, Greider CW, Villeponteau B. The rna component synthesis using thymidine incorporation. of human telomerase. Science 1995; 269: 1236–1241. The effects of cytotoxic agents on telomerase activity have 4 Greider CW. Telomere length regulation. Annu Rev Biochem 1996; 65: 337–365. been scarcely documented to date. Most of them reduced 5 Zhu XL, Kumar R, Mandal M, Sharma N, Sharma HW, Dhingra telomerase activity, as seen with inhibitors of reverse tran- U, Sokoloski JA, Hsiao RS, Narayanan R. Cell cycle-dependent scriptase,26 tubulin poisons that induce an M-phase block, modulation of telomerase activity in tumor cells. Proc Natl Acad such as taxol and nocodazole,5,22 antimetabolites that induce Sci USA 1996; 93: 6091–6095. an S-phase block such as methotrexate and 5-fluorouracil,5,22 6 Holt SE, Wright WE, Shay JW. Regulation of telomerase activity 22 in immortal cell lines. Mol Cell Biol 1996; 16: 2932–2939. and cytotoxic agents that induce G2 arrest, such as cisplatin, 5 7 Kramer KM, Haber JE. New telomeres in yeast are initiated with and topoisomerase inhibitors (although not always, see a highly selected subset of TG1-3 repeats. Genes Dev 1993; 7: Ref. 27). There are only a few reports of telomerase up- 2345–2356. regulation: after irradiation (the present study), at the onset of 8 Flint J, Craddock CF, Villegas A, Bentley DP, Williams HP, Gal- apoptosis,22 an in response to high doses of hydroxyurea, anello R, Cao A, Wood WG, Ayyub H, Higgs DR. Healing of which includes DNA fragmentation when applied for long broken human chromosomes by the addition of telomeric repeats. periods of time.5,28 Therefore, since telomerase activation has Am J Hum Genet 1994; 55: 505–512. 9 Broccoli D, Young JW, de Lange T. Telomerase activity in normal only been detected subsequent to double-strand DNA breaks, and malignant hematopoietic cells. Proc Natl Acad Sci USA 1995; a putative role for telomerase in their repair is possible. 92: 9082–9086. Few reports suggest telomerase involvement either in the 10 Nilsson P, Mehle C, Remes K, Roos G. Telomerase activity in vivo protection of DNA from degradation or directly in DNA in human malignant hematopoietic cells. Oncogene 1994; 9: repair. The results of two studies support a role for telomerase 3043–3048. in DNA protection from degradation. In the first, telomere 11 Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, Shay JW, Ishioka S, Yamakido M. Activation of telomerase length in human cells that lack telomerase was reported to in human lymphocytes and hematopoietic progenitor cells. J shorten at a 10-fold faster rate than expected from incomplete Immunol 1995; 155: 3711–3715. replication, suggesting that telomeric DNA is degraded by 12 Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S. Telomerase nucleases.29 In the second, a reduction in dicentric formation activity in normal leukocytes and in hematologic malignancies. was observed following irradiation of ataxia telangiectasia Blood 1995; 85: 2315–2320. fibroblasts expressing telomerase, compared with fibroblasts 13 Shay JW, Werbin H, Wright WE. Telomeres and telomerase in 30 human leukemias. Leukemia 1996; 10: 1255–1261. that had no telomerase activity. Telomerase involvement in 14 Clave E, Carosella E, Gluckman E, Dubray B, Socie G. 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