Induction of telomerase activity by UV-irradiation in Chinese ... - Nature

Oncogene (1997) 15, 1747 ± 1752  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

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Induction of telomerase activity by UV-irradiation in Chinese hamster cells M Prakash Hande1, AS Balajee2 and AT Natarajan MGC-Department of Radiation Genetics and Chemical Mutagenesis, Leiden University, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands

Telomerase is a ribonucleoprotein whose activity has been detected in germline cells and in neoplastic and immortal cells. Telomerase compensates the telomere loss arising by the end replication problem by synthesizing telomeric repeats at the 3' end of the eukaryotic chromosomes. Telomerase is reactivated during cancer progression in human and mice. In order to determine whether the telomerase activity can be upregulated in vitro in response to DNA damaging agents, we examined the telomerase activity in ®ve Chinese hamster cell lines following exposure to 5 J/m2 or 40 J/m2 UV-C radiation. All the cell lines tested showed an increase in telomerase activity in the PCRbased telomeric repeat ampli®cation protocol (TRAP) in a dose dependent manner. This increase in telomerase activity correlated well with the number of cells being in the S and G2/M phase after UV exposure. However, in unirradiated control cells, similar levels of telomerase activity were observed in dierent phases of the cell cycle. Furthermore, telomeric signals were clustered in one or more parts of the disintegrating nuclear particles of the apoptotic cell as detected by ¯uorescence in situ hybridization (FISH). This is the ®rst study to demonstrate the induction of telomerase activity following exposure to DNA-damaging agents like UV radiation in Chinese hamster cells in vitro. Keywords: telomeres; Hamster cells

telomerase;

UV

radiation;

Telomeres comprise of tandem arrays of (TTAGGG)n repeats in most eukaryotes (Moyzis et al., 1988). Telomeres serve as protective caps that prevent chromosomes from fusing together and causing DNA rearrangements that can lead to karyotypic changes and genomic instability (Greider, 1991). Actively dividing cells both in vivo and in vitro show a progressive decline in telomeric length after every replicative cycle because of the end replication problem (Allsopp et al., 1995). Telomere shortening has been proposed as a regulatory mechanism that controls the number of times a cell can divide before undergoing cellular senescence, thereby acting as mitotic clock (Harley, 1991). In immortal cells

Correspondence: MP Hande or AT Natarajan Present addresses: 1The Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver V5Z 1L3 Canada; 2 Laboratory for Molecular Genetics, Gerontology Research Center, National Institute of Aging, 4940 Eastern Avenue, Baltimore, Maryland 21224 USA Received 3 February 1997; revised 2 June 1997; accepted 3 June 1997

including cancer cells, telomere shortening has been circumvented by reactivation of the enzyme telomerase, a ribonucleoprotein. Telomerase adds short repetitive telomeric sequences to the chromosome termini thus stabilizing the telomere length and allowing the cells to divide inde®nitely (Greider and Blackburn, 1985; Morin, 1989). Telomerase activity has been reported in germline cells and in immortalized cells, but it has not been detected in normal adult somatic tissues or cultured human diploid cells with exception of renewal tissues that contain stem cells (Kim et al., 1994; Broccoli et al., 1995; Hiyama et al., 1995; Wright et al., 1996). Telomerase activity has been shown to be upregulated in a variety of tumor cells and tumor growth is associated with telomerase activity (Counter et al., 1994, 1995; Kim et al., 1994; Chadeneau et al., 1995a, Bacchetti, 1996). However, the precise mechanism for the telomerase reactivation is not clearly understood. Recently telomerase activity has been detected in the skin of sun exposed individuals suggestive of the fact that UV-B-irradiation alone could trigger the telomerase activity (Taylor et al., 1996; Ueda et al., 1997). In order to determine whether primary DNA lesions produced by UV-C-irradiation could induce the telomerase activity, we have undertaken studies in immortalized Chinese hamster cells which exhibit telomerase activity. Five spontaneously immortalized Chinese hamster cell lines, namely lung ®broblasts (CHL), embryonic ®broblasts (CHE), V79, CHO9 (Chinese hamster ovary) and radiation sensitive mutant cell line, xrs5 (kindly provided by Dr PA Jeggo, MRC Cell Mutation Unit, Brighton, UK) were exposed to 5 J/ m2 or 40 J/m2 with a Philips T.U.V. lamp (predominantly 254 nm) at a dose rate 0.2 J/m2/s. Control groups were treated similarly except for the exposure to UV. Exposed as well as unexposed cells were allowed to grow for another 20 h during which they complete one division and harvested for TRAP assay, ¯ow cytometric analysis and for FISH analysis on interphase cells. All the cell lines showed easily detectable telomerase activity (Figure 1a, lanes representing control groups). Expression of telomerase in these immortalized cells is in conformation with the fact that immortalized cells express telomerase activity (Kim et al., 1994). Semiquantitative analysis of the bands in the autoradiograms was done by Phosphorimager (Molecular Dynamics, USA) and using the ImageQuant software (Molecular Dynamics, USA). Telomerase activity was upregulated as compared to the unirradiated control cells (Figure 1a and b). The degree of increase was dependent on the dose of UV employed. This is

Induction of telomerase activity MP Hande et al

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a

40 J/m2

5 J/m2

Control

V79 40 J/m2

5 J/m2

CHE Control

40 J/m2

5 J/m2

CHO9 Control

40 J/m2

Control

40 J/m2

5 J/m2

Control

5 J/m2

xrs5

CHL

ITAS

b

Figure 1 (a) Autoradiograph showing telomerase bands in dierent Chinese hamster cells in control and irradiated groups. Telomerase activity was measured by TRAP assay using an endlabeled telomerase substrate (TS) primer as described (Kim et al., 1994). Cell extracts were prepared by lysing the cells in CHAPS buer, centrifuged at 14 000 r.p.m. at 48C for 45 min and 2 ml (approximately 2000 cells) of these extracts were used in the assay. The telomerase reaction was carried out in 50 ml of TRAP buer containing 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween-20, 1 mM EGTA, 50 mM of deoxynucleotide triphosphate, 0.1 mg of the labeled TS primer, ACX primer, U2 primer 561073 attamoles of an internal control primer (TSU2) and 2 units of Taq DNA polymerase (BoehringerMannheim) and 2 ml of CHAPS extract. Reaction tubes were placed in a robocycler (Stratagene, USA) for 30 min at 308C, followed by 27 cycles of PCR at 948C for 30 s and 728C for 30 s. About 30 ml of the ampli®ed products were resolved on 12% polyacrylamide gel, dried and visualized by autoradiography using BioMax ®lms after 12 h of exposure at room temperature. Extracts treated with RNase gave negative results (data not shown). (b) Telomerase activity in dierent Chinese hamster cell lines after U.V.-C-irradiation. For semiquantitative analysis of telomerase activity, gels were exposed overnight to phosphor screens at room temperature and visualized on a PhosphorImager using ImageQuant software (Molecular Dynamics, USA). The signals from individual extract were normalized for the band obtained for internal standard (ITAS). The relative telomerase activity was determined by calculating the ratio of the intensity of the area under bands representing telomeric repeats (TRAP ladder) to the area under the band representing ampli®ed internal standard. Initially TRAP assay was done according to Kim et al. (1994) without using an internal standard and TRAP products were quanti®ed by computerized scanning densitometry using a Hewlett Packard Scanjet IIIC. The semiquantitative analysis was performed on a Macintosh computer using the public domain NIH image program (developed at the National Institute of Health and available on the Internet at http://rsb.info.nih.gov/ nih-image/). Relative telomerase activity obtained after normalizing for the amount of protein used in PCR showed similar tendency in the increase in the enzyme activity

perhaps the ®rst study to show modulation of telomerase activity in vitro by a DNA-damaging agent, i.e. UV radiation. It has been reported that telomerase activity is activated during tumorigenesis in transgenic animals (Chadeneau et al., 1995b); Blasco et al., 1996; Broccoli et al., 1996) and is upregulated during radiation-induced malignant transformation of human cells (Pandita et al., 1996). More recently, Taylor et al. (1996) and Ueda et al. (1997) have found higher levels of telomerase in the sun-exposed skin compared to unexposed skin in humans indicating a possible modulation of telomerase activity by U.V. exposure. Activation of telomerase in the skin is associated with chronic sun exposure and occurs at an early stage of carcinogenesis (Ueda et al., 1997). There were no reports available so far demonstrating the induction of telomerase activity after treatment with DNA-damaging agents in vitro. Clearly, there was a 2 ± 4-fold increase in telomerase activity in the UV exposed hamster cells. We believe that this elevation of telomerase activity could be due to the chromosome breakage events that occur after UV exposure and stabilization of this damage by a mechanism for chromosome healing by telomerase. Under normal circumstances, telomere shortening due to the end replication problem is probably sensed by telomerase as a strand break and (TTAGGG)n repeats are added by telomerase to maintain the telomeric length. An analogous mechanism may be involved for the detection of strand breaks induced by DNA-damaging agents. The induction of DNA (single and double) strand breaks by UV, as a consequence of ongoing excision repair of photolesions, are recognized by telomerase for addition of telomeric repeats at these sites. A chromosome arm not terminating in a telomere is prone to fusion and recombination and can undergo breakage-fusion-bridge cycles (Kipling, 1995). It is known that double strand breaks can also activate checkpoint controls and cause cell cycle arrest. Therefore broken chromosome should be healed for the normal cell cycle progression. Naturally occurring chromosomal truncations or chromosome breakage by integration of arti®cial constructs are healed by telomerase in human cell lines (Barnett et al., 1993; Murnane and Yu, 1993; Flint et al., 1994). Breakage within or close to, interstitial telomeric repeats in hamster cells may also serve to stabilize induced chromosome breaks by providing a substrate for telomerase. Balajee et al. (1996) have reported that interstitial telomeric sequences are prone to DNA damage because of their sequence composition and chromosomal location. The presence of free ends of chromosomes following exposure to DNA-damaging agents facilitates telomerase to use them as primers for the addition of telomeric repeats both in vivo and in vitro (Flint et al., 1994). New telomeres may be seeded following breakage within the intrachromosomal telomeric repeats to stabilize the rearranged chromosomes (Bertoni et al., 1994). As there was a dose dependent increase in telomerase activity, it is likely that the extent of DNA damage might trigger the telomerase activity and the UV-doses used in the present experiment are high enough to induce sucient amounts of breakage events. Though telomerase is already present in these immortalized cells, it appears from our results that this level may not be sucient to


maintain the stability of the chromosomes following DNA damage. Thus, additional telomerase activity may be necessary to stabilize the induced chromosomal breakage sites. X-ray induced chromosomal aberration data support this hypothesis (Slijepcevic et al., 1996) as telomeric capping was observed at breakage site only after a S phase.

Flow cytometric data revealed that in the three cell lines, CHO9, V79 and xrs5, the induction of telomerase activity parallels the number of cells in S phase and G2/M phase at the time of extraction especially at the high UV-dose used (Figure 2a ± e). The other two cell lines, CHE and CHL, showed such an increase only at the 40 J/m2 UV-dose. Recently,

Figure 2 FACS pro®les showing percentage of cells at dierent phases of cell cycle in irradiated and unirradiated Chinese hamster cells. Cell cycle analysis was done by uptake and intercalation of propidium iodide (PI) to DNA. Cells were processed by using a detergent-trypsin method (Vindelov et al., 1983) with minor modi®cations as follows. Cells were incubated for 10 min at room temperature with 450 ml of 0.003% trypsin in detergent solution (3.4 mM Trisodium Citrate. 2H2O, 1% v/v Non idet P40, 1.5 mM Spermine tetrachloride, 0.5 mM Tris pH, 7.6). Then 375 ml of detergent solution with trypsin inhibitor (0.5 g/L) and RNase A (0.1 g/ L) were added to the cells and incubated for 10 min at room temperature. Finally, a third incubation at room temperature was carried out for at least 15 min with 375 ml of detergent solution containing PI (0.42 g/L) and Spermine tetrachloride (1.2 g/L). The cell suspensions were kept on ice until used for ¯owcytometric analysis. About 10 000 cells were analysed with a FACSCAN ¯owcytometer (Becton and Dickinson, USA). Analysis of the distribution of percentage of cells in dierent phases of cell cycle according to cell DNA content was performed on gated nuclei using the software program (CELLquest) provided by Becton Dickinson. M1, M2 and M3 represent G1, S and G2/M phases of the cell cycle respectively. The percentages of cells in dierent phases of cell cycle are given with the FACS pro®le for each group. (a) V79 cells, (b) CHE cells, (c) CHO9 cells, (d) CHL cells and (e) xrs5 cells

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Figure 3 FISH with telomeric DNA on xrs5 interphase cells. Interphase cells were hybridized with telomeric DNA. Cells were harvested, ®xed in methanol:acetic acid (5 : 1) solution and dropped on a clean slide. The telomeric DNA probe was generated by PCR following the protocol of Ijdo et al. (1991). PCR was carried out in the absence of template using primers (TTAGGG)5 and (CCCTAA)5. Staggered annealing of primers provided single stranded template for extension by Taq polymerase. Ampli®cation consisted of ®rst 10 cycles (1 min at 948C, 30 s at 608C and 90 s at 728C) followed by 30 cycles (1 min at 948C, 30 s at 608C and 90 s at 728C). PCR-ampli®ed DNA was found as a smear in agarose (0.8%) consisting of fragments ranging from 2 to 25 kb in length. An amount of 1 mg of PCRampli®ed DNA was labeled with biotin-16 dUTP by nick translation. The slides were incubated with pepsin (0.005%) in 10 mM HCl for 10 min at 378C. After washing with PBS containing 50 mM MgCl2, the slides were post®xed with 1% formaldehyde in PBS/MgCl2 for 10 min at room temperature. They were washed with PBS and dehydrated in a 70, 90 and 100% ethanol series. The biotinylated probe was diluted with hybridization buer to 2 ng/ml). Diluted probe (20 ml) was added on each slide, under a 24650 mm coverslip. The slides and the probe were heat denatured at 808C for 4 min and hybridized overnight at 378C in a humidi®ed chamber. After hybridization, the slides were washed three times with 50% formamide and 26SSC at 428C. For immunological detection, the slides were incubated with avidin-FITC (Vector laboratories, USA) for 30 min at room temperature. The signal was ampli®ed using biotinylated goat-anti-avidin antibody (Vector labs). After dehydration with ethanol series, the slides were counterstained with PI and DAPI (Sigma) mixed with antifade Vectashield (Vector Labs) mounting medium. Fluorescence microscopy was performed on a Zeiss Axioplan microscope (Carl Zeiss Germany) equipped with ®lters for observation of DAPI (blue), FITC (green), TRITC (red) and a triple ®lter for simultaneous observation of DAPI, FITC and TRITC. Color images were collected with a cooled CCD camera (Photometrics USA) and were processed with IP-Lab software in a Power PC Apple computer. The distribution of signals in the interphase cells were observed with the FITC ®lter. Using the DAPI ®lter the slides were scanned for apoptotic cells. (a) xrs5 interphase cells of control group showing telomeric signals. (b) xrs5 interphase cells after treatment with UV at a dose of 40 J/m2 showing strong and ampli®ed signals in the nuclei. (c) Cells treated with UV at a dose of 40 J/m2 showing an apoptotic cell which can be clearly

Holt et al. (1996) reported that telomerase is active throughout the cell cycle in dividing, immortal cells but its activity is repressed in G0 cells. However, Zhu et al. (1996) reported that human tumor cells arrested at G1/S phase of the cell cycle showed similar levels of telomerase activity to asynchronous cultures, but progression through the S phase was associated with increased activity. To see whether there is any dierential expression of telomerase during dierent phases of cell cycle, cells were assayed at dierent time points after collecting the cells by mitotic shake o method. It was found that all the dierent phases of cell cycle in actively dividing cells expressed similar levels of telomerase (data not shown) suggesting that telomerase activity is not regulated in a cell cycle phase-dependent manner. Cell cycle regulation of telomerase activity was not detected in human cells as well (Holt et al., 1996). In the present investigation, we studied the telomerase activity only during the proliferative stages of the cell cycle. Though similar levels of telomerase activity during dierent phases of cell cycle supports the observation that the elevation in telomerase activity may be due to DNA damage induced by UV-irradiation, we cannot rule out the possibility that the elevated activity might also be due to the dierences in the proportion of cells that are cycling. Telomerase could also be directly activated by UV exposure through mitotic proliferation of telomerase positive cells. Further investigations are required to study the mechanism by which the induction of

Table 1

V79 Control 5 J/m2 40 J/m2 CHE Control 5 J/m2 40 J/m2 CHL Control 5 J/m2 40 J/m2 CHO9 Control 5 J/m2 40 J/m2 xrs5 Control 5 J/m2 40 J/m2

Apoptotic cells in Chinese hamster cells following UVirradiation Cells scored

Apoptotic cells

Percent

Telomere clusters

Percent

3000 3000 3000

0 19 128

0 0.6 4.3

0 6 82

0 31.0 64.1

3000 3000 3000

0 33 35

0 1.1 1.2

0 21 22

0 63.6 62.9

3000 3000 3000

0 67 15

0 2.2 0.5

0 48 13

0 71.6 86.7

3000 3000 3000

0 27 83

0 0.9 2.8

0 13 51

0 48.1 61.4

3000 3000 3000

0 96 261

0 3.2 8.7

0 65 186

0 67.7 71.0

Interphase cells were scored after FISH with telomere probe (See legends of Figure 3). Apoptotic cells were scored under DAPI ®lter and telomere signals were qualitatively observed under the FITC ®lter

observed under a DAPI ®lter. Apoptosis was characterized as rounded apoptotic bodies or fragmented nucleus that contained dense chromatin masses. (d) The same cell as c under FITC ®lter showing telomere clusters in the disintegrating nuclear bodies of the apoptotic cell. (e) Another cell showing strong signals in the interphase nuclei and telomere clustering in apoptotic cells after UV-irradiation at 40 J/m2


telomerase activity occurs in vitro following exposure to DNA-damaging agents and its in¯uence on malignant transformation. The observation of telomerase induction by UVirradiation in Chinese hamster cells raises questions as to what is the exact role of this elevation in these cells. It is of interest to determine whether telomeric DNA is ampli®ed by telomerase in UV treated cells. Therefore, we next examined the signal intensities of telomeric DNA after FISH with telomeric probe in interphase cells following UV treatment. Though FISH signal intensities in the interphase cells, observed qualitatively, seem to be stronger after UV exposure (Figure 3a and b), it is unlikely that telomerase can add sequences/repeats within one cell division. Novel quantitative FISH would be useful in the measurement of telomeric signals before and after exposure to environmental agents. More interestingly, it was observed that telomeric signals were clustered to one or more parts of the nucleus in apoptotic cells with bright intensities (Figure 3c ± e). The data on the apoptotic cells observed after UV-irradiation and telomeric signal intensities in the apoptotic cells are presented in Table 1. The percentage of apoptotic cells was increased after UV exposure in these Chinese hamster cells which is dependent on the dose employed. In the X-ray sensitive mutant cell line, xrs5, the eect was pronounced. About 60 ± 85% of the apoptotic cells showed strong signals in one or more speci®c disintegrating nuclear bodies (Figure 3c ± e, Table 1). It is possible that UV-induced strand breaks are healed by telomeric repeats which are added by the telomerase. This healing process may lead to chromosomal instability, as these repeats are added randomly at various chromosome sites, thus triggering the cells to undergo apoptosis. Though this should be explored further in the future studies, premature chromosome

instability has also been observed in human ®broblasts after irradiation with high LET particles (Sabatier et al., 1995). In chromosomally unstable lymphoblastoid cells, high frequency of apoptotic cells has been reported as compared to normal cells (Duchaud et al., 1996). Telomerase induction by UV-irradiation in immortalized Chinese hamster cells as seen in the present study, is the ®rst example to show that exposure to DNA-damaging agents may upregulate the enzyme activity. We have also observed the induction of telomerase activity following exposure to ionizing radiation (X-rays) in human tumor cell lines in vitro (Oh et al., 1997) as well as in mouse splenocytes in vivo (Hande and Natarajan, manuscript in preparation). The induction of telomerase activity following exposure to DNA-damaging agents must be investigated more thoroughly to determine whether similar mechanisms occur during cancer progression and whether increased level of telomerase has any in¯uence on the telomere length regulation. Studies are underway to see whether prerequisite of telomerase activity is necessary for the elevation of the enzyme following exposure to DNA-damaging agents in vitro.

Acknowledgements We thank JC Kluin-Nelemans and A van der Marel, Department of Haematology, Academisch Ziekenhuis Leiden for their assistance in ¯ow cytometry; S Sebastian, A Errami and J Yui for help during the study. Thanks are due to Peter M Lansdorp for allowing one of the authors (MPH) to use some of the facilities at the Terry Fox Laboratory. This work was ®nancially supported by a CEC Nuclear Fission Safety grant to ATN.

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