Upregulation of telomerase activity by X-irradiation in mouse ...

Carcinogenesis vol.21 no.4 pp.573–578, 2000

Upregulation of telomerase activity by X-irradiation in mouse leukaemia cells is independent of Tert, Terc, Tnks and Myc transcription

Paul Finnon, Andrew R.J.Silver and Simon D.Bouffler1 Radiation Effects Department, National Radiological Protection Board, Chilton, Didcot, Oxon OX11 ORQ, UK 1To

whom correspondence should be addressed Email: [email protected]

X-irradiation of two mouse myeloid leukaemia cell lines was found to lead to increased telomerase activities. Maximal increases in activity at 24 h post-irradiation were approximately three times control unirradiated cell levels. These maxima were reached at between 3–5 Gy depending upon cell line. Peak activity was reached at 8 h, remained elevated to 24 h and returned to control levels by 48 h. In contrast, X-irradiation did not activate telomerase in a telomerasenegative human fibroblast line, while in cultured normal mouse bone marrow cells irradiation appeared to reduce activities. No simple relationship between radiation-induced increases in telomerase activity in the myeloid leukaemia lines and the proportions of cells in the S or M phases of the cell cycle was apparent. Radiation-induced increases in activity were significantly reduced by inhibitors of transcription (actinomycin D, α-amanatin) and protein synthesis (cycloheximide). These data are consistent with two possibilities: (i) X-irradiation leads to increased transcription and/or translation of a component of telomerase, thus increasing activities; or (ii) X-irradiation induces the transcription of a positive regulator of telomerase activity. Northern blot analysis did not indicate that transcription of mTert, the catalytic subunit of telomerase, or mTerc, the RNA component, was elevated after irradiation. Similarly, no significant changes in the expression of Myc or Tnks, the tankyrase gene, two suspected telomerase regulators, were detected. These data are therefore consistent with the induction by X-irradiation of a positive regulator of telomerase activity other than Tnks or Myc or the core protein and RNA components of the enzyme.

Introduction Telomerase is a ribonucleoprotein enzyme which directs the synthesis of telomeric DNA repeats from an RNA template (reviewed in 1). In human somatic cells, telomerase activity is not readily detected except in tissues with strong self renewal capacity, suggesting that active enzyme is present in stem cells or proliferating precursor cells (2–5). In contrast, most human tumour samples express high levels of telomerase activity (2,3,6–8). Many cancers, including leukaemias, are thought to originate from stem or primitive progenitor cells (9–11). Thus, telomerase may be involved in carcinogenesis at early stages. In mouse, telomerase activity has been observed in both normal somatic tissues and tumour cells with higher levels generally in the tumour cells (12,13). Thus, mouse cells provide an ideal system in which to study the regulation of telomerase following carcinogenic insult. © National Radiological Protection Board

The acquisition of telomerase activity during immortalization of human cells counters telomere shortening and the often associated tendency for chromosomes to fuse (14). Absence of telomerase in mice lacking telomerase RNA (mTerc–/– mice) leads to progressive telomere shortening and compromised proliferation in germ line (testes) and haemopoietic (bone marrow and spleen) tissues, these defects are most apparent in animals bred for several generations without active telomerase (15,16). Telomerase also contributes to genome stability as seen by increased occurrence of aneuploidy and Robertsonian fusion in cells from late generation mTerc–/– mice (15,16). In addition to acting at the ends of chromosomes, telomerase can heal broken chromosomes at non-telomeric locations, the best examples in higher eukaryotes being constitutive chromosome 16 truncations in α-thalasaemia patients (17,18). A review has suggested that telomerase healing of radiation-induced chromosome breaks is a relatively rare event in mammalian cells (19). Nevertheless only very short homologies to the canonical telomere repeat appear to be required to direct telomerase healing (18). Double-stranded DNA breaks induced by ionizing radiation may therefore present an abundant substrate for healing by telomerase. Telomerase healing is unlikely to act on all breaks as multiple pathways of repair of double-stranded breaks exist in mammalian cells (20). These multiple pathways may be regarded as being in competition for the processing of chromosome breaks. In support of a role for telomerase in the response of cells to DNA damage it has been reported that telomerase activity can be elevated following exposure of Chinese hamster cells to shortwave UV light (21) and human haemopoietic and colon tumour cell lines to γ-irradiation (22,23). In this paper, it is reported that X-irradiation transiently increases telomerase activities in two mouse myeloid leukaemia cell lines in a dosedependent fashion. These increases were significantly reduced when cells were incubated in the presence of inhibitors of RNA or protein synthesis. Induced activity increases were not paralleled by elevated transcription of mTert, the telomerase catalytic subunit which is considered to be a major regulator of telomerase activity (24–26). The RNA component of telomerase has also been implicated in regulating telomerase activity (27–30) but again, here no elevation of mTerc levels was observed following irradiation. Radiation exposure did not elevate levels of expression of two other known or suspected telomerase regulators, Myc (31,32) and Tnks, the tankyrase gene (33). These results are therefore consistent with the hypothesis that X-irradiation induces the transcription of a telomerase-positive regulator protein distinct from Myc and tankyrase. Materials and methods Cells Mouse radiation-induced myeloid leukaemia cell lines mlp3 (strain of origin CBA/H, gift from Dr E.Meijne, ECN, Petten, The Netherlands) and 8709 (strain of origin C3H, gift from Dr K.Yoshida, NIRS Chiba, Japan) were maintained in RPMI 1640 (Gibco BRL) supplemented with 10% fetal calf

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P.Finnon, A.R.J.Silver and S.D.Bouffler serum (Life Technologies), 100 IU/ml penicillin, 100 µg/ml streptomycin and 2 mM glutamine at 37°C with a 5% CO2/air atmosphere. Normal human fibroblast strain MA85 (gift from Dr D.Scott, The Paterson Institute, Manchester, UK) was grown in Minimal Essential Medium (Life Technologies) supplemented with 15% fetal calf serum and 2 mM glutamine. Normal mouse bone marrow cells were extracted from CBA/H mice. Cells were grown in RPMI 1640 plus 20% fetal calf serum supplemented with 30% WEHI cell conditioned medium. Irradiation conditions Cells were seeded into T25 or T75 flasks 1 day prior to irradiation to achieve log phase growth at time of irradiation. Cultures were irradiated with 250 kVp X-rays at a dose rate of 0.73 Gy/min. Telomerase activity determination At time of sampling, cell number and viability were checked by haemocytometer counts in the presence of trypan blue. After washing in PBS, 106 viable cells were resuspended in 200 µl CHAPS lysis buffer (10 mM Tris–HCl, 1 mM MgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 0.1 mM PMSF, 5 mM β-mercaptoethanol) and incubated for 30 min on ice. Lysates were then spun at 13 000 r.p.m. for 30 min at 4°C in a benchtop ultracentrifuge (Beckman), the supernatant collected and stored at –70°C. Protein concentrations were determined using the Coomassie blue reaction (Bio-Rad). Telomerase activities in lysates were determined with a telomeric repeat amplification protocol (TRAP) assay kit (TRAP-eze, Oncor Appligene) following the manufacturer’s protocol with the exception that 50 µl PCRs included 1 µg T4 gene 32 protein (Boehringer Mannheim) and 2 µCi 3000 Ci/mmol [α-32P]dCTP, primers were not end labelled. Extract sample volumes were adjusted for equal protein or equal viable cell number input. PCRs were run for 30 cycles of 94°C for 30 s, 55°C for 30 s and products run on 10% non-denaturing polyacrylamide gels at 275–300 V for 2–3 h in 0.5⫻ TBE. Gels were fixed for 30 min in 30% ethanol, 0.5 M NaCl, 0.04 M sodium acetate and dried onto 3 MM paper. Gels were exposed to X-ray film (Kodak Biomax MR) for varying periods for autoradiography. TRAP assay products were quantitated by scanning densitometry (Bio-Rad GS700 imaging densitometer) of autoradiograms exposed within the linear response range of the film. Intensity of the first product band and all the product bands was assessed and normalized to the short internal control (36 bp) band intensity. Activity in experimental samples was expressed relative to control unirradiated samples. RNA–protein synthesis inhibitors Actinomycin D, cycloheximide and α-amanatin (Sigma) were used at 10 µg/ml, 100 µg/ml and 10–8 M, respectively. Inhibitors were present for 30 min prior to irradiation and for the 5 h prior to cell lysis. Cell cycle analysis Cells were plated at 2⫻105/ml 16–24 h prior to irradiation/sampling. At the appropriate time bromodeoxyuridine (BrdU; Sigma) was added at a final concentration of 50 µM for 30 min. Aliquots of cells were transferred to slides by cytocentrifuge (Shandon) and fixed in 3:1 methanol:acetic acid for 15–30 min. Cells which incorporated BrdU (i.e. S-phase cells) were detected immunologically. Non-specific antibody binding was blocked by a 30 min incubation in 4⫻ SSC, 5% non-fat dried milk, 0.05% Tween-20. Slides were incubated for 45 min at 37°C in a 1:250 dilution of mouse monoclonal anti-BrdU antibody (clone BU-33, Sigma). This was followed by three washes in 4⫻ SSC 0.5% Triton X-100, 45 min incubation at 37°C in 1:100 sheep anti-mouse IgG fluorescein isothiocyanate (FITC) conjugate (Sigma), another wash series, dehydration through 70, 90 and 100% ethanol and air drying. Slides were mounted in Vectashield (Vector Labs) containing 100 ng/ml 6-diaminophenylindole dihydrochloride and 500 µg/ml propidium iodide. Slides were observed under a Nikon epi-fluorescence microscope equipped with appropriate filter sets. S-phase cells were recognized by bright FITC (yellow/green) fluorescence and mitotic cells by their morphology. The number of S- and M-phase cells in randomly selected samples of 250–500 cells was determined. Gene expression studies Aliquots of 107 cells were lysed in 4 M guanidium isothiocyanate, 50 mM Tris–HCl pH 7.5, 25 mM EDTA. Total RNA was then extracted using an RNeasy kit (Qiagen), DNase I digested and further purified through an RNeasy column. Aliquots (20 µg) of total RNA were separated on 1.5% agarose gels containing formaldehyde, transferred to Hybond N membranes (Amersham) by northern blotting. Blots were probed sequentially with 25 ng β-actin, mTerc, myc and Tnks cDNA probes 32P-labelled using a Rediprime kit (Amersham) and washed to a stringency of 0.5⫻ SSC at 42°C. Blots were autoradiographed and the signals quantitated similarly to TRAP assay gels (see above). cDNAs were produced by random hexamer (Boehringer Mannheim) primed reverse transcription using Superscript II (Life Technologies). The control

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Fig. 1. Time-course of telomerase activity increase following 3 Gy X-irradiation of mlp3 cells. (a) TRAP activity gel autoradiograph, postirradiation sample times are given above the appropriate lanes. PCRs were assembled with 200 ng cell extract protein per reaction H2O, water blank; CHAPS, CHAPS extraction buffer control; TSR8, positive reaction control primer set; IC, internal 36 bp control. (b) Quantitation of autoradiograph. Relative telomerase activity represents the TRAP assay product yield assessed by scanning densitometry normalized to the internal control band (IC) intensity divided by the TRAP assay product yield of the normalized unirradiated control. β-actin probe was generated by PCR amplification of CBA/H cDNA using a commercial β-actin positive control primer set (Stratagene). The mTERT probe was generated by nested RT–PCR of 8709 cDNA initially with primers F1/R2 (5⬘-CGTCGATACTGGCAGATGCGG-3⬘; 5⬘-CATGTTCATCTAGCGGAAGGAGACAG-3⬘). The product of the first reaction was then amplified with primers F3/R3 (5⬘-TCCAACAGCTGCTGGTGAACCA-3⬘; 5⬘-GAAGGAGACAGGGTTAGTCC-3⬘) generating a 2189 bp probe between nucleotides 1222 and 3411 (numbering according to GenBank accession no. AF051911). The myc probe was generated by RT–PCR of 8709 RNA with primers 5⬘-CCAACAGGAACTATGACCTC-3⬘; 5⬘-TTGCTCTTCTTCAGAGTCGC-3⬘ to produce a 745 bp fragment between nucleotides 629 and 1374 [according to Hann et al. (34); GenBank accession no. X01023]. The Tnks probe was produced by RT–PCR of normal human fibroblast RNA using primers based on the human sequence (33; GenBank accession no. AF082556). The primers used (5⬘-TCAGCCAATTTCTAAAAAGCC-3⬘; 5⬘-TTCTGCTCTGCGGCTGTTGC-3⬘) produced an 885 bp fragment spanning nucleotides 3097–3982. This probe hybridized to two transcripts (~4.5 and 2 kb) in normal human fibroblast line MA85 (data not shown) and similarly sized transcripts were detected in the mouse cell lines (Figure 6). A probe for mTerc was produced by RT–PCR using primers 5⬘-GGTCGAGGGCGGCTAGGCTC-3⬘; 5⬘-GGTGCACTTCCCACAGCTCAG-3⬘ A probe running from nucleotides 65 to 465 was produced (numbering according to GenBank accession no. U33831).

Results Telomerase activity is increased following irradiation TRAP assays of extracts of mouse acute myeloid leukaemia cell lines mlp3 and 8709 revealed all were positive for telomerase activity (Figures 1a and 2). X-irradiation (3 Gy) of

X-ray induction of telomerase activity

Fig. 3. Quantitation of X-ray dose–responses for telomerase activity changes in mlp3, 8709 and normal bone marrow cells. Relative telomerase activity (y-axis, relative to appropriate unirradiated control) was determined by scanning densitometry as stated in Materials and methods. For normal bone marrow activities are expressed relative to unirradiated WEHI-stimulated cell activity levels. X-ray doses are given on the x-axis. TRAP PCRs were assembled using extract from 104 viable cells. Fig. 2. Dose–response for telomerase activity increases in 8709 cells. Autoradiograph of TRAP assay products using extract from 104 viable cells harvested 24 h after irradiation. X-ray doses are given above each lane.

mlp3 cells transiently increased telomerase activity (Figure 1a). Scanning densitometry of the autoradiographs allowed quantitation of the radiation effect, the time-course (Figure 1b) is plotted in terms of relative telomerase activity, i.e. activity in the irradiated samples divided by the activity in control, unirradiated samples. A relatively rapid increase in activity to ~2-fold unirradiated control levels was observed, this increase was maintained to 24 h post irradiation (Figure 1b). The increase decayed to control levels by 48 h and lower by 72 h (Figure 1). The decline below control levels may be due, in part, to cell killing. Dose–responses were determined for the cell lines at the fixed time point of 24 h post-irradiation. In this experiment TRAP assays were assembled using extract aliquots from equal numbers of viable cells. This served to maximize the increases detected. Figure 2 shows TRAP assay product yields in control and irradiated 8709 cells. Increasing X-ray dose gave greater product yield, i.e. greater telomerase activity. Quantitation of autoradiographs from assays of 8709 and mlp3 (Figure 3) showed a clear dose–response for both cell lines but each line had its own characteristics. Maximal activities reached their peaks at ~3-fold control, unirradiated values at between 3 and 5 Gy. As well as increasing the yield of smaller TRAP assay products, extracts from irradiated cells produced a larger range of products (Figure 2). This suggests that increased enzyme processivity may be in part responsible for the increased activity. Normal mouse bone marrow cells grown in WEHI cellconditioned medium were found to have detectable telomerase activity, albeit at an ~12-fold lower level than the myeloid leukaemia cell lines (data not shown). Furthermore, the activity in WEHI-stimulated cells was ~3-fold greater than that in fresh unstimulated bone marrow cells. Irradiation of WEHIstimulated bone marrow cell cultures tended to reduce rather than increase activities, although no simple dose dependency was evident (Figure 3). Irradiation of a telomerase activity negative normal human fibroblast strain (MA85) did not activate the enzyme to detectable levels (data not shown).

Fig. 4. S-phase index (left panel, y-axis) and mitotic index (right panel, y-axis) in mlp3 (stars) and 8709 (circles) at 24 h after a range of X-ray doses from 1 to 5 Gy (x-axis).

Cell cycle perturbation Some reports in the literature indicate that telomerase activity in mammals is cell cycle stage dependent (35–37) with highest activities occurring during S phase; however, other investigators fail to observe such regulation (22,38,39). In view of the possibility of cell cycle stage dependent regulation and the widely observed phenomenon of radiation-induced changes in cell cycle distribution, S- and M-phase indices in irradiated cultures were checked. As expected, irradiation reduced the proportions of cells in S- and M-phase in both cell lines (Figure 4). There was no obvious direct or inverse correlation between telomerase activity and proportion of Sor M-phase cells. The cell cycle responses to radiation do, however, differ substantially in the two cell lines, mlp3 cells responded to X-irradiation with less effective blocks to S- and M-phase entry than 8709 cells (Figure 4). Inhibitor studies As a first step in investigating the mechanism of telomerase activity upregulation by radiation, experiments with protein/ RNA synthesis inhibitors were carried out. In all experiments, the inhibitor(s) were added to cultures 30 min prior to irradiation and remained present during irradiation and for the 575

P.Finnon, A.R.J.Silver and S.D.Bouffler

Fig. 5. Inhibitors of RNA and protein synthesis block the X-ray induced increase in telomerase activity in 8709. The presence (⫹) or absence (–) of the inhibitors actinomycin D and cycloheximide is indicated below each bar. Inhibitors were present for 30 min prior to 3 Gy X-irradiation and for a 5 h post-irradiation period after which extracts were prepared. TRAP PCRs were assembled with extract from 104 viable cells. Relative telomerase activities were determined from TRAP assay product gels.

subsequent 5 h culture period. The presence of 100 µg/ml cycloheximide in unirradiated cultures had a minimal effect on telomerase activities which were 90–120% of control untreated levels. This agrees broadly with Holt et al. (39) who found that telomerase activity has a long (⬎24 h) half-life in most cells. However, cycloheximide eliminated any radiation effect on telomerase activity in 8709 (Figure 5). If the elevation of telomerase activity was due to effects on protein stability or post-translational mechanisms, cycloheximide would not block the response to radiation. The effect of 10 µg/ml actinomycin D on unirradiated cultures was more variable with activities between 50 and 90% of control levels. This inhibitor blocked the upregulation of telomerase activity in 8709 (Figure 5). Unirradiated, α-amanitin-treated mlp3 cultures had telomerase activities of 84–104% control levels; again, this inhibitor blocked the elevation of telomerase activity by radiation (data not shown). Expression of telomerase components and regulators The inhibitor studies suggested that the elevation of telomerase activity following exposure to radiation was mediated by the transcription and translation of a protein. A strong candidate for this protein is mTert which is known to be a key regulator of telomerase activity (24–26). Northern blot analysis of RNAs extracted from unirradiated cells and cells exposed to 3 Gy X-rays 1, 3, 8 and 24 h previously did not reveal any significant alteration in mTert RNA levels by radiation in mlp3 (Figure 6) or 8709 (data not shown). No significant elevation of the mTert RNA level in mlp3 was detected following irradiation either. Thus, neither of the two core telomerase components appears to be implicated in the radiation response. Myc has been shown to regulate telomerase (31,32). In mlp3, myc mRNA levels did appear somewhat elevated at 8 h after 3 Gy X-irradiation, but otherwise remained at control unirradiated cell levels (Figure 6). The expression of the final suspected telomerase regulator, Tnks (33), was not significantly altered by 3 Gy X-irradiation at the times examined. Discussion Telomerase activity is commonly detected in human cancers (2,3,6–8) and the acquisition of an effective telomere mainten576

Fig. 6. Effect of X-irradiation on known regulators of telomerase activity. RNA samples were prepared from mlp3 1, 3, 8 or 24 h after 3 Gy X-irradiation or unirradiated control cells (indicated above lanes), run on formaldehyde gels and northern blotted. The blot was sequentially probed with mTERT, myc, Tnks and β-actin gene probes. Note heavier loading of 8 and 24 h samples in β-actin panel.

ance mechanism is considered to be a key step in human carcinogenesis (40,41). While the majority of human somatic cells have no detectable telomerase activity, stem cells or primitive progenitor cells, in particular those of haemopoietic origin, have been found to be positive for telomerase activity (3–5). In common with the findings in human cell lines (22,23), telomerase activities in two mouse myeloid leukaemia cells were found to be transiently elevated following exposure to low–moderate doses of X-irradiation (Figures 1–3). This induction appeared to be specific to the myeloid leukaemia cells as no increases were detected in cultured normal bone marrow or a normal human fibroblast line. Clearly the regulation of telomerase in these cell types is different. In vitro exposure of hamster cells to UV-irradiation has also been found to increase telomerase activity (21); similarly, normal human skin at chronically sun-exposed locations frequently shows activity (42). However, no effect of UV was seen in a nasopharyngeal cell line (43). A variety of chemical carcinogens (3⬘-methyl-4-dimethyl-aminoazobeneze, aflatoxin B1, methyl nitrosourethane, dimethylnitrosamine, diethylnitrosamine) have been found to elevate telomerase activity in rat tissues (44). In contrast, doxorubicin, bleomycin, methotrexate, melphalan and transplatin had no effect on human testicular tumour cell telomerase activities (45), whereas cisplatin reduced activity (45). Again, these chemical agent findings are controversial as Ku et al. (43) observed no effects of cisplatin, methyl methane sulfonate or two topoisomerase II inhibitors. Here, as in two other studies (21,22), the increased activity observed in mouse myeloid leukaemia cell lines did not appear

X-ray induction of telomerase activity

to be caused by shifts in cell cycle distribution or increased proliferation (Figure 4). The effect therefore appeared to be a genuine response to the radiation treatment. Thus, in some situations where telomerase is expressed it is subject to regulation by DNA damaging agents and may therefore play a part in the cellular response to DNA damage, so possibly affecting the outcome of therapeutic treatment of some cancers. In the case of ionizing radiation, transient elevation of telomerase activity is a consistent finding (22,23; this study). We observed no reactivation of telomerase in a telomerasenegative human fibroblast line and radiation-induced reductions in activity in short-term cultured mouse bone marrow cells. Thus, normal somatic cells and primary cell strains appear to behave differently to tumour cell lines and permanent cell lines. This suggests at least two levels for the regulation of telomerase exist, initial expression of activity and then modulation of that activity. This modulation can be in a positive or negative fashion. To investigate the mechanism of telomerase activity elevation, transcription/translation inhibitor experiments and northern blot analysis were undertaken. The inhibitor studies reported here had two aims. Firstly to determine whether the radiation-associated increases in telomerase activity were due to regulation at a transcriptional, translational or posttranslational level. The time course of the elevated activity (Figure 1) could be compatible with control at any of these levels. Secondly, the use of cycloheximide, the inhibitor of translation, would reveal whether a protein or an RNA component were involved if regulation were at the transcriptional level. Current evidence suggests that telomerase activity in mice and humans is most commonly regulated by levels of hTERT/mTert, the reverse transcriptase subunit (25,26,46) rather than the RNA components, hTER/mTerc (47–50). The modulation of telomerase activity by ionizing radiation reported here is inhibitable by both transcriptional and translational inhibitors. Therefore, the regulation appears to be through the transcription of a protein component. Furthermore, northern blot analysis did not reveal any correlation between mTerc levels and activity (Figure 6). The expression level of mTert was examined and found to be not significantly altered by radiation in any cell line or at any time point examined (Figure 6 and data not shown). Thus, the observed radiation-induced elevation of telomerase activity appears not to be effected by mTert. The involvement of mTerc, the telomerase RNA component can also be excluded as the response is blocked by the protein synthesis inhibitor cycloheximide and no correlation between activity and mTerc transcript levels was observed. Thus, these data imply that radiation induces the transcription of a telomerase activity stimulating protein in mouse leukaemic cells. Myc has recently been identified as an activator of telomerase in human cells (31,32). A possible increase in Myc transcript levels was detected in mlp3 at 8 h after radiation exposure; this, however, does not correlate well with the telomerase activity increases. This is perhaps not surprising as Myc appears to act through the transcriptional activation of Tert (31,32) and no increase in Tert transcript levels was observed here. Another recently identified candidate is tankyrase, a poly(ADP-ribose) polymerase located at human telomeres identified by Smith et al. (33) who speculate that telomerase activity might be modulated by tankyrase-mediated ADP-ribosylation. However, the ubiquitous expression of Tnks (33) argues against its involvement in the radiation response described here and,

furthermore, no effect of radiation on Tnks expression was observed (Figure 6). Additionally, the spatial restriction of tankyrase to telomeres (33) perhaps suggests that if tankyrase does act as a telomerase activity regulator, it will do so in a local rather than global fashion. A number of studies have indicated that telomerase activity can be influenced at the post-translational level. Li et al. (51) report the specific inhibition of nuclear telomerase activity in extracts by protein phosphatase 2A (PP2A), protein phosphatases I and IIB were ineffective. Furthermore, okadaic acid, a PP2A inhibitor, was found to stimulate telomerase activity in a human breast cancer cell line (51). In contrast, it is claimed that an inhibitor of PP1γ can inhibit telomerase activity in a PP2A-negative human myeloid leukaemia cell line (52). From these data it is unclear whether phosphorylation increases or decreases telomerase activity either directly or indirectly. In support of the possibility that phosphorylation of telomerase or a telomerase regulator positively regulates activity, Ku et al. (43) report that protein kinase C inhibitors inhibit telomerase activity in a human nasopharyngeal cancer cell line. A protein kinase A inhibitor and protein tyrosine kinase inhibitors had no effect (43). None of these studies of telomerase activity modulation has addressed whether the effect is direct or indirect. It is quite possible that telomerase is subject to a variety of levels of regulation and indeed different regulatory mechanisms might operate in different cellular systems. In summary, in the present study it was found that Xirradiation of two mouse leukaemia cell lines transiently elevated telomerase activities ~3-fold. The involvement of mTerc and mTert in this increased activity has been excluded. Similarly Myc and Tnks expression appears not to be involved. The data presented are consistent with the hypothesis that radiation induces the transcription of an as yet unidentified positive regulator of telomerase. Further studies are needed to identify this regulator and to elucidate the role of telomerase in the response to DNA damage. Acknowledgements We are very grateful to Drs Emmy Meijne, Kazuko Yoshida and David Scott for providing cell lines. We also thank John Moody for help in preparing the figures, Kathy Brooks for typing the manuscript and Dr Roger Cox for useful discussions. The MRC Radiation and Genome Stability Unit is acknowledged for provision of X-irradiation facilities. This work was supported in part by the European Commission under contract F14-PCT-950008.

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