Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity

Home , DNA ligase, DNA replication, Ku70, Protein subunit, Reverse transcriptase, Telomerase

Vol. 10, 2551–2560, April 1, 2004 Clinical Cancer Research 2551

Ki-Hyuk Shin,1 Mo K. Kang,1 Erica Dicterow,1 INTRODUCTION Ayako Kameta,1 Marcel A. Baluda,1 and Telomerase, which consists of the catalytic protein subunit, No-Hee Park1,2 human telomerase reverse transcriptase (hTERT), the RNA component of telomerase (hTR), and several associated pro- 1School of Dentistry and 2Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California teins, has been primarily associated with maintaining the integ- rity of cellular DNA telomeres in normal cells (1, 2). Telomer- ase activity is correlated with the expression of hTERT, but not ABSTRACT with that of hTR (3, 4). Purpose: From numerous reports on proteins involved The involvement of DNA repair proteins in telomere main- in DNA repair and telomere maintenance that physically tenance has been well documented (5–8). In eukaryotic cells, associate with human telomerase reverse transcriptase nonhomologous end-joining requires a DNA ligase and the (hTERT), we inferred that hTERT/telomerase might play a DNA-activated protein kinase, which is recruited to the DNA role in DNA repair. We investigated this possibility in nor- ends by the DNA-binding protein Ku. Ku binds to hTERT mal human oral fibroblasts (NHOF) with and without ec- without the need for telomeric DNA or hTR (9), binds the topic expression of hTERT/telomerase. telomere repeat-binding proteins TRF1 (10) and TRF2 (11), and Experimental Design: To study the effect of hTERT/ is thought to regulate the access of telomerase to telomere DNA telomerase on DNA repair, we examined the mutation fre- ends (12, 13). The RAD50, MRE11, and NBS1 proteins, which quency rate, host cell reactivation rate, nucleotide excision are involved in DNA repair, are also active in telomere elonga- repair capacity, and DNA end-joining activity of NHOF and tion via protein kinase ATM and associate with TRF1 and TRF2 NHOF capable of expressing hTERT/telomerase (NHOF-T). (13–17). Moreover, recent observations indicate that telomerase NHOF-T was obtained by transfecting NHOF with hTERT also associates with proteins known to participate in DNA plasmid. replication (18) and in DNA repair (9). Also, DNA repair is Results: Compared with parental NHOF and NHOF impaired in mice with telomere dysfunction (19, 20). Because transfected with empty vector (NHOF-EV), we found that proteins involved in DNA replication are required for repair of a) the N-methyl-N؅-nitro-N-nitrosoguanidine-induced mu- damaged DNA, there exists a possibility that telomerase is also) tation frequency of an exogenous shuttle vector was reduced involved in DNA repair. A recent report showed that ectopic in NHOF-T, (b) the host cell reactivation rate of N-methyl- expression of hTERT accelerated the repair of DNA double- N؅-nitro-N-nitrosoguanidine-damaged plasmids was signifi- strand breaks induced by ionizing radiation and of DNA adducts cantly faster in NHOF-T; (c) the nucleotide excision repair produced by cisplatin (21). of UV-damaged DNA in NHOF-T was faster, and (d) the To investigate the putative role of hTERT in general DNA DNA end-joining capacity in NHOF-T was enhanced. We repair, two independent strains of normal human oral fibroblasts also found that the above enhanced DNA repair activities in (NHOF) were transfected with a plasmid capable of expressing NHOF-T disappeared when the cells lost the capacity to hTERT. NHOF do not express the hTERT gene, which is express hTERT/telomerase. silenced by hypermethylation (22). They express hTR, which is Conclusions: These results indicated that hTERT/te- ubiquitously present in normal cells. Three NHOF clones that lomerase enhances DNA repair activities in NHOF. We expressed hTERT and telomerase activity were established. hypothesize that hTERT/telomerase accelerates DNA repair Two of the three clones transiently expressed telomerase activ- by recruiting DNA repair proteins to the damaged DNA ity because the nonintegrated plasmids were lost after approxi- sites. mately 35 population doublings (PDs) after transfection. During the period when these hTERT-transfected clones expressed hTERT and possessed telomerase activity, they demonstrated a significantly greater DNA repair capacity compared with that of the parental NHOF and NHOF transfected with empty vector Received 5/1/03; revised 12/29/03; accepted 12/31/03. (NHOF-EV). After the hTERT-transfected cells ceased to ex- Grant support: Grants DE14147 and DE14635 funded by the National press hTERT and telomerase activity, they lost the capacity to Institute of Dental and Craniofacial Research. The costs of publication of this article were defrayed in part by the enhance DNA repair. The hTERT-induced enhancement of payment of page charges. This article must therefore be hereby marked DNA repair was demonstrated in four different ways. First, in advertisement in accordance with 18 U.S.C. Section 1734 solely to NHOF expressing telomerase activity treated with N-methyl-NЈ- indicate this fact. nitro-N-nitrosoguanidine (MNNG), the mutation frequency of Requests for reprints: No-Hee Park, University of California Los Angeles School of Dentistry, CHS 53-038, 10833 Le Conte Avenue, the replicating pS189 shuttle vector was decreased. Second, Los Angeles, CA 90095-1668. Phone: (310) 206-6063; Fax: (310) 794- NHOF expressing telomerase activity had a higher repair level 7734; E-mail: [email protected]. of exogenous DNA damaged in vitro by MNNG. Third, NHOF

expressing telomerase activity showed a faster rate of nucleotide protein. The PCR products were electrophoresed in 12.5% non- excision repair (NER) in both strands of an endogenous cellular denaturing polyacrylamide gels, and the radioactive signals gene. Fourth, NHOF expressing telomerase activity had a higher were detected by PhosphorImager (Molecular Dynamics, Inc., rate of DNA end-joining activity. The parental primary NHOF Sunnyvale, CA). and five hTERT-negative clones served as controls. Southern Blot Analysis. For genomic Southern blot analysis, 10 ␮g of DNA were digested with SalI restriction enzyme. The fragmented DNA was then separated by electro- MATERIALS AND METHODS phoresis, transferred to nitrocellulose filter, and hybridized to Cells and Culture Conditions. Primary cultures of 32P-labeled full-length hTERT cDNA. The hTERT cDNA was NHOF were established from explants of gingival connective labeled with [32P]dCTP (ICN Radiochemicals, Irvine, CA) by tissue that was excised from patients undergoing oral surgery. Prime-It RmT Random Primer Labeling Kit (Stratagene). The The cells that proliferated outwardly from the explant culture radioactive signals were detected by PhosphorImager and quan- were continuously cultured in 100-mm culture dished in titatively analyzed by ImageQuant software (Molecular Dynam- DMEM/medium 199 (4:1) containing fetal bovine serum (Gem- ics Inc., Sunnyvale, CA). ini Bioproducts) and gentamicin (50 ␮g/ml). Primary normal Analysis of Mutation Frequency in pS189 Shuttle Vec- human oral keratinocytes were prepared from separated epithe- tor. The shuttle vector pS189 and host Escherichia coli strain lial tissue and serially subcultured in Keratinocyte Growth Me- MBM7070 obtained from Dr. E. J. Shillitoe (State University of 2ϩ dium (Clonetics) containing 0.15 mM Ca as described previ- New York, Syracuse, NY) were described elsewhere (25, 26). ously (23). 293 (adenovirus-transformed human embryonic The pS189 vector can replicate in a number of different cell kidney) cells were obtained from American Type Culture Col- types, including lymphoblasts (24), cells from xeroderma lection (Manassas, VA) and cultured in DMEM/medium 199 complementation groups (27), normal oral human keratinocytes, (4:1) containing fetal bovine serum (Gemini Bioproducts) and and oral squamous cell carcinoma cells (26, 28). Eighty percent gentamicin (50 ␮g/ml). To determine cell PDs, the cells were confluent cells were transiently transfected with pS189 plasmids subcultured until they reached postmitotic stage. PD of the cells using LipofectAMINE 2000 reagent (Invitrogen) as described was calculated at the end of each passage by the formulation 2N previously. Twenty-four h after transfection, the cells were ϭ (Cf/Ci), where N denotes PD, Cf denotes the total cell number exposed to 1.5 ␮g/ml MNNG for 2 h and cultured in fresh harvested at the end of a passage, and Ci denotes the total cell medium not containing the chemical for an additional 24 h. At number of attached cells at seeding. The PD time was calculated the end of the incubation, the plasmids were recovered from the by dividing the duration of culture in hours by the PD value. cells by the alkaline lysis method as described elsewhere (29). Transfection and Cloning. A human TERT expression Plasmid DNA was digested with the enzyme DpnI (Boehringer plasmid (pCI-neo-hTERT) was provided by Dr. Robert A. Mannheim Biochemicals, Indianapolis, IN) to cleave DNA from Weinberg (Whitehead Institute for Biomedical Research, Mas- plasmids that had not replicated in NHOF (30). Recovered sachusetts Institute of Technology). Control plasmid (pCI-neo) plasmid DNA that had replicated in cells were transfected into was obtained from Promega (Madison, WI). After 41 PDs, E. coli MBM7070 by the heat shock method. Transformed approximately 2 ϫ 105 exponentially replicating NHOFs per bacteria were plated on Luria-Bertani agar plates containing 60-mm culture dish were transfected with pCI-neo-hTERT or ampicillin (50 ␮g/ml), 5-bromo-4-chloro-3-indolyl-␤-D-galacto- pCI-neo by using LipofectAMINE 2000 reagent (Invitrogen). pyranoside, and isopropyl-1-thio-␤-D-galactopyranoside. Bacte- For each 60-mm dish, a mixture of pCI-neo-hTERT or pCI-neo rial colonies containing plasmids with the mutant or wild-type (5 ␮g/50 ␮l) and the LipofectAMINE reagent (35 ␮g/50 ␮l) was suppressor tRNA gene (supF) were identified by color (cells added to the culture medium dropwise as uniformly as possible containing wild-type plasmids are blue, whereas cells with mu- with gentle swirling. The cells were incubated for 7 h at 37°C. tant plasmids are light blue or white). The mutagenic frequency The medium was then replaced with fresh culture medium, and was determined as the percentage of white and light blue colo- the cultures were incubated for an additional 24 h. To select nies in the total colonies [mutagenesis frequency (%) ϭ number cells transfected with the pCI-neo-hTERT or pCI-neo, the cells of white and light blue colonies Ϭ number of total colonies]. were incubated in culture medium containing 200 ␮g/ml G418 Host Cell Reactivation Assay. The pGL3-Luc plasmid (Invitrogen). Then, G418-resistant clones were isolated by ring- (Promega), in which expression of the firefly luciferase gene is cloning. The G418-resistant clones transfected with pCI-neo- controlled by the cytomegalovirus (CMV) promoter, was used hTERT or pCI-neo were selected and subcultured. to determine the capacity of cells for repairing damaged DNA. Nucleic Acid Isolation. High molecular weight cellular The pRL-CMV plasmid, in which the Renilla luciferase gene is DNA was extracted from the cells with phenol/chloroform/ driven by the CMV promoter, was used as an internal control for isoamyl alcohol (25:24:1) and ethanol precipitation (24). transfection efficiency. To create in vitro damaged DNA, the Analysis of Telomerase Activity. Cellular extracts were pGL3-Luc plasmid was exposed to 50 or 100 ng/ml MNNG for prepared by using 3-[(3-cholamidopropyl)dimethylammonio]-1- 30 min in Tris-EDTA buffer and purified with Wizard DNA propanesulfonic acid (lysis buffer) provided from the TRAP-eze Clean-Up System (Promega). Approximately 5 ϫ 104 cells/well Telomerase Detection Kit (Intergen Corp., Norcross, GA) as were plated in a 24-well culture dish and cultured for 24 h. The recommended by the manufacturer. Telomerase activity was cells were then transiently transfected with 1 ␮g damaged determined using the TRAP-eze Telomerase Detection Kit as pGL3-Luc plasmid/well and 0.1 ␮g pRL CMV plasmid/well described previously (23). Each telomeric repeat amplification using the LipofectAMINE reagent following the manufacturer’s protocol reaction contained cellular extract equivalent to 1 ␮g instructions. The pRL-CMV plasmid was used to normalize for

total DNA transfected. After4hoftransfection, the transfection nonhomologous end-joining activity that precisely rejoins bro- medium was replaced with regular culture medium. Cells were ken DNA ends in vivo. The pGL3 plasmid was completely collected 48 h after transfection, and cell lysates were prepared linearized by restriction endonuclease NarI (New England Bio- according to the Promega’s instruction manual. Luciferase ac- labs), which cleaves within the luciferase coding region as tivity was measured using the Dual Luciferase Reporter Assay confirmed by agarose gel electrophoresis. The linearized DNA System (Promega) and a luminometer (Promega). The Renilla was subjected to phenol/chloroform extraction and ethanol pre- luciferase activity was used to normalize for transfection effi- cipitation and dissolved in sterilized water. ciency. Before transfection, a 6-well plate was inoculated with NER Assay. Strand-specific riboprobes for the p53 approximately 5 ϫ 104 cells/well and cultured for 24 h. The EcoRI fragment detection were prepared by PCR amplification cells were then transiently transfected with 1 ␮g linearized of a human genomic DNA fragment between nucleotides 1750 pGL3 plasmid/well or 1 ␮g intact pGL3 plasmid/well using the and 2138 of exon II of the p53 gene. This fragment of 388 LipofectAMINE reagent (Invitrogen) following the manufactur- nucleotides was ligated into the pGEM-T plasmid (Promega) er’s instructions. After7hoftransfection, the transfection and sequenced to confirm the absence of mutations. Strand- medium was replaced with regular culture medium. Cells were 32 specific P-labeled riboprobes were generated using the T7 or collected 48 h after transfection, and cell lysates were prepared SP6 transcription promoters in the pGEM-T/p53 vector as de- according to the Promega instruction manual. Luciferase activ- scribed previously (31). Strand-specific removal of UV-induced ity was measured using the Luciferase Reporter Assay System cyclobutane pyramidine dimers (CPDs) was analyzed from a (Promega) and a luminometer (Promega). The reporter plasmid 16-kb EcoRI fragment of the active p53 gene using single- was digested to completion with NarI within the luciferase stranded labeled riboprobes specific for p53 (32, 33). Cells coding region, and only precise DNA end-joining activity cultured to confluence were UV-irradiated with 2.5 J/m2 (254 should restore the luciferase activity. The precise nonhomolo- nm). Genomic DNA was extracted at 0, 8, and 24 h after gous end-joining activity was calculated from the luciferase UV-irradiation using phenol/chloroform/isoamyl alcohol (25: activity of linearized pGL3 plasmids compared with that of the 24:1) and ethanol precipitation (24). The extracted DNA was uncut plasmids. Each experiment was repeated three times. digested with EcoRI and treated or mock-treated with T4 endonuclease V (T4V; Epicenter Technologies, Madison, WI) and then electrophoresed on denaturing agarose gels and transferred RESULTS to Hybond-N nylon membranes (Amersham Life Sciences, Ar- Expression of hTERT Induces Telomerase Activity in lington Heights, IL). After hybridization with strand-specific Two Independent Strains of NHOF. Two actively prolifer- riboprobes, signals in the membrane were detected using a ating independent strains of NHOF (NHOF-1 at PD 41 and Storm 840 phosphorimager and quantified with ImageQuant NHOF-2 at PD 37) were transfected with a plasmid (pCI-neo- software, version 1.2 (Molecular Dynamics). Repair of CPDs hTERT) capable of expressing hTERT and neomycin phospho- was calculated by comparing the amount of radioactivity in the transferase. Fifteen G418-resistant colonies of NHOF-1 and 12 T4V-treated versus mock-treated fragments and normalized colonies of NHOF-2 were cloned and tested for telomerase with a loaded control plasmid (pGL3 control plasmid). activity by the telomeric repeat amplification protocol assay. In Vitro DNA End-Joining Assay. Cells were collected Among the fifteen G418-resistant cell clones of NHOF-1, two and washed three times in ice-cold PBS. The cells were lysed by incubation for 30 min at 4°C in lysis buffer [1% Triton X-100, clones expressed telomerase activity when tested at PD 50 (clones FT-1 and FT-3). The parental NHOF and a control clone 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA (Fneo), NHOF transfected with pCI-neo without hTERT cDNA, (pH 8.0), 0.2 mM sodium orthovanadate, and protease inhibitor mixture (Boehringer Mannheim)]. The cell lysates were centri- did not display telomerase activity (Fig. 1, A and B). The fuged at 8000 ϫ g for 10 min at 4°C. EcoRI linearized pCR2.1- telomerase-positive NHOF clones were serially subcultured to TOPO plasmid (Invitrogen) was incubated with total cell ex- determine the effect of telomerase on replication and senescence tracts for2hat37°Cin20␮l of reaction mixture containing 1 of NHOF. Also, telomerase activity was tested again in FT-1 at ␮l of linearized plasmid (10 ng), 2 ␮l of cell extract (10 ␮g), 4 PDs 62, 74, and 84 and in FT-3 at PDs 62, 76, and 86 (Fig. 1, ␮l of 50% polyethylene glycol, and 2 ␮lof10ϫ ligase buffer A and B). At PDs 62 and 74, FT-1 showed telomerase activity, [300 mM Tris-HCl (pH 7.8), 100 mM KC1, 100 mM DTT, and 10 which was decreased by 5.5-fold in cells at PD 74. A similar mM ATP]. To amplify rejoined DNA, PCR reaction was per- pattern of reduced telomerase activity was noted in FT-3 at formed with 3 ␮l of end-joining reaction using M13 reverse higher PDs. Telomerase activity was not detected in FT-1 and primer (5Ј-CAGGAAACAGCTATGAC- 3Ј) and M13 forward FT-3 at PD 84 and PD 86, respectively, although these clones primer (5Ј-GTAAAA CGACGGCCAG-3Ј). The PCR condition continued to replicate exponentially (Fig. 1, A and B). FT-3 consisted of 35 cycles at 95°C for 30 s, 60°C for 30 s, and 70°C expressed approximately four times less telomerase activity than for 30 s. PCR products were separated in 2% agarose gel FT-1 at all times tested. Thus, telomerase activity was only electrophoresis in Tris-borate EDTA buffer and visualized by transiently expressed in the hTERT-transfected NHOF clones. staining with ethidium bromide. Amplification of rejoined DNA The 12 G418-resistant cell clones established from was evident as a 186-bp band. NHOF-2 were also tested for hTERT expression by the telo- In Vivo DNA End-Joining Assay. The pGL3 plasmid meric repeat amplification protocol assay. Among the 12 G418- (Promega), in which expression of the luciferase gene is con- resistant cell clones, 1 clone (T-11) expressed telomerase activ- trolled by the CMV promoter, was used to evaluate correct ity (Fig. 1C). The parental NHOF and three clones transfected

Fig. 1 Expression of exogenous telomerase activity in two independent strains of normal human oral fibroblasts (NHOF). A, telomeric repeat amplification protocol assay of parental, vector-transfected (Fneo), and human telomerase reverse transcriptase-transfected NHOF (FT-1 and FT-3). Cellular extracts after different population doubling were tested for telomerase activity. The positive controls were 293 cells (adenovirus-transformed human embryonic kidney cells) and normal human oral keratinocytes (NHOK). The 3-[(3-cholamidopropyl)- dimethylammonio]-1-propanesulfonic acid buffer was used as a negative control. B, quantification of telomerase activity. Signals in the gel were detected using a Storm 840 PhosphorImager and quantified with ImageQuant software, version 1.2 (Molecular Dynamics). The level of telomerase activity was normalized to the level of an internal control amplification product. C, telomeric repeat amplification protocol assay with another strain of NHOF transfected with the empty vector (N-1, N-2, and N-p) or the human telomerase reverse transcriptase-expressing vector (T- series). Only T-11 clone expressed telomerase activity. IC, internal control amplification product.

with the empty vector (N-1, N-2, and N-p) did not display transiently transfected into parental NHOF (PD 60), Fneo (PD telomerase activity. 62), FT-1 (PD 65), and FT-3 (PD 65). The luciferase activity hTERT/Telomerase Decreases the Mutation Frequency was monitored in all tested cells. The hTERT-expressing fibro- of a Shuttle Vector in NHOF Treated with a DNA-Damaging blasts demonstrated a significantly higher level of luciferase Agent. To investigate the effect of telomerase on DNA repair, we compared the mutation frequency of pS189 shuttle vector plasmids (25, 34) in cells unexposed or exposed to the genotoxic agent MNNG. Because FT-1 and FT-3 cells showed a decreased Table 1 Spontaneous and MNNGa-induced mutation frequencies of expression of telomerase activity at PD 74 and a total loss of pS189 shuttle vector in the control and hTERT-transfected activity at PD 84 (Fig. 1), we used cells at PD 65. The sponta- oral fibroblasts neous mutation frequency (1/23,049) of the pS189 plasmid in NHOF were transfected with pS189 plasmids, exposed to MNNG the clones FT-1 and FT-3 was similar to that (1/28,131) in for 2 h, and incubated in fresh medium for 24 h. The pS189 plasmids were recovered and introduced into E. coli MBM7070. Mutagenesis parental NHOF and in Fneo analyzed at PD 60 and PD 62, assay was performed as described previously (26). Cells at PD 65 were respectively during the exponential replication phase (Table 1). used for the assay. After the cells were treated with MNNG, the mutation frequency Plasmid recovered of the shuttle vector was significantly increased in all of the tested cells. However, the magnitude of the increase was almost no. of mutants/no. Mutagenesis ␮ two times lower in FT-1 and FT-3 cells than that in the parental Cells MNNG ( g/ml) of total colonies frequency (%) and Fneo cells (Table 1). These data indicated that the expres- NHOF 0 0/12705 0 sion of hTERT/telomerase activity either prevented the mutation 1.5 1350/14305 9.4 Fneo 0 1/15426 0.006 of the plasmids or increased the repair of DNA damaged by 1.5 1931/17993 10.7 MNNG. FT-1 0 0/12189 0 hTERT/Telomerase Enhances the Repair of MNNG- 1.5 759/17613 4.3b Damaged Exogenous DNA. The host cell reactivation assay FT-3 0 1/10860 0.009 1.5 746/11789 6.3b is a method of investigating the DNA repair capacity of cells by a quantifying the function of repaired exogenous DNA that had MNNG, N-methyl-NЈ-nitro-N-nitrosoguanidine; hTERT, human telomerase reverse transcriptase; NHOF, normal human oral keratino- been damaged before introduction into cells (35–37). cytes; PD, population doubling. The pGL3-Luc luciferase reporter plasmids were treated b Significantly lower than the mutation frequency in NHOF and with 0, 50, or 100 ng/ml MNNG. The damaged plasmids were Fneo exposed to MNNG (1.5 ␮g/ml; ␹2 test).

cellular p53 gene was analyzed using a single-stranded 32P- labeled riboprobe specific for either the transcribed or nontranscribed strand of p53 (Fig. 3). In the parental NHOF, within 8 h after UV-irradiation, 33% of the transcribed and 20% of the nontranscribed DNA strands were repaired, and within 24 h after UV-irradiation, 56% of the transcribed and 38% of the nontranscribed strands were repaired. In Fneo, the percentages of repair were 36% and 24% after 8 h and 68% and 44% after 24 h, respectively, for the two DNA strands. In the hTERT/ telomerase-expressing cells, the NER activity was much faster. In the FT-1 clone, within 8 h after irradiation, 60% of the transcribed and 46% of the nontranscribed DNA strands were repaired, and within 24 h after UV-irradiation, 100% of the transcribed and 65% of the nontranscribed DNA strands were repaired. In the FT-3 clone, the repair percentages for the two DNA strands were 44% and 31% after 8 h and 90% and 45% Fig. 2 Faster host cell reactivation rate in normal human oral fibro- after 24 h. The slower rate of NER repair in FT-3 than in FT-1 blasts (NHOF) capable of expressing human telomerase reverse tran- appears to reflect the lower expression of telomerase activity scriptase (hTERT)/telomerase. A, quantification of luciferase activity repaired by host cell reactivation. Parental NHOF, vector-transfected resulting from fewer active hTERT plasmids in FT-3. The delay NHOF (Fneo), and hTERT-transfected NHOF (FT-1 and FT-3) were in repairing the nontranscribed strand was not affected by te- transfected with either undamaged or N-methyl-NЈ-nitro-N-nitrosogua- lomerase activity. The NER assay was not performed with the nidine (MNNG)-damaged pGL3-Luc reporter plasmid for 4 h. After T-11 clone. Ⅺ 48 h, cell lysates were prepared and luciferase activity was measured. , hTERT/Telomerase Increases the DNA End-Joining luciferase activity of undamaged plasmids; u, luciferase activity of plasmids damaged by 50 ng/ml MNNG; f, luciferase activity of plas- Activity in NHOF. DNA end-joining is part of the mecha- mids damaged by 100 ng/ml MNNG. The error bars indicate ϮSD from nism for repairing double-strand DNA breaks (41, 42). To P Ͻ 0.05 versus controls. B, host cell reacti- determine whether hTERT/telomerase is directly involved with ,ء .three different assays vation assay of luciferase activity in empty vector-transfected clones double-strand breaks repair, we carried out an in vitro end- (N-1, N-2, and N-p) and in hTERT-transfected fibroblast clone express- P Ͻ 0.05 versus controls (N-1, N-2, joining assay using linearized plasmid and PCR amplification of ,ء .(ing telomerase activity (T-11 and N-p). rejoined DNA (Fig. 4A). The cellular extracts from FT-1 and FT-3 expressing telomerase activity at PD 62 showed 50–70% higher end-joining activity than that of Fneo at PD 69 within a 2-h incubation period (Fig. 4B). An additional control clone was activity compared with the controls. At the higher (100 ng/ml) also examined in this experiment. One of the 13 hTERT-trans- concentration of MNNG, the level was twice as high as in fected clones, FT-5, which failed to demonstrate telomerase controls. The luciferase activity of undamaged plasmids was activity, showed end-joining activity similar to that of the con- similar in the controls and the hTERT-transfected cells (Fig. trol (Fig. 4, A and B). 2A). In the in vivo DNA end-joining assay, the three control Similar results were also obtained from the independent clones with empty vector (N- 1, N-2, and N-p) showed only hTERT transfection study with the NHOF-2 strain. The three 58% of the DNA repair activity detected in the hTERT-express- clones transfected with empty vector (N-1, N-2, and N-p) ing clone (Fig. 4C). These data confirmed the results obtained showed only 55% of the host cell reactivation activity detected from the hTERT-transfected NHOF-1 strain and indicated that in the hTERT-expressing clone (T-11; Fig. 2B). hTERT/telomerase expression was associated with the enhance- hTERT/Telomerase Accelerates the NER Process of ment of in vitro and in vivo DNA end-joining activity in NHOF. Cellular Gene. In mammalian cells, a variety of DNA lesions Enhanced DNA Repair Activity Disappears When the such as UV-induced CPDs are repaired by the NER pathway Cells Lost Telomerase Activity. We also compared the rep- (38). The NER pathway is divided into transcription coupled lication capacity of the hTERT-transfected NHOF-1 clones repair, which is restricted to the transcribed strand of transcrip- (FT-1 and FT-3) with that of the parental cells and the empty tionally active genes, and general genome repair, which acts on vector-transfected cells (Fig. 5A). The parental cells and the DNA lesions within the entire genome (38, 39). vector-transfected control ceased dividing at PD 82 and PD 80, The effect of hTERT/telomerase on NER of the endoge- respectively, whereas the hTERT-transfected clones, FT-1 and nous p53 gene was measured by the rate of removal of UV- FT-3 replicated for an additional 20 and 10 doublings, respec- induced CPDs from the individual strands of the gene sequence tively. The T-11 clone is replicating at present, and the cells (40). This assay involves both transcribing strand-specific NER have not yet reached senescence. and general NER. The parental, vector-transfected, and hTERT/ To determine whether the loss of telomerase activity and telomerase-expressing fibroblasts at PD 65 were irradiated with cellular replication arrest were caused by arrest of hTERT 2.5 J/m2, and cellular DNA was isolated 0, 8, and 24 h after expression or by the loss of nonintegrated hTERT carrying UV-irradiation. The extracted DNA was treated with T4V, plasmids, we investigated the physical status of the exogenous which cleaves DNA containing UV-induced CPDs. The strand- hTERT gene by Southern blot analysis of cellular DNA isolated specific removal of CPDs from a 16-kb EcoRI fragment of the from clones FT-1 and FT-3 (Fig. 5B). The cellular DNA was

Fig. 3 Acceleration of nucleotide excision repair (NER) by telomerase activity. NER assay in parental, vector-transfected, and human telomerase reverse transcriptase-transfected fibroblasts. Strand-specific removal of UV-induced cyclobutane pyrimidine dimers from a 16-kb EcoRI fragment of the active p53 gene using a specific single-stranded ␣-32P-labeled riboprobes. DNA was extracted from the cells 8 and 24 h after UV- irradiation (hpUV), digested with EcoRI, and treated (ϩ) or mock treated (Ϫ) with T4 endonuclease V (T4V). DNA was electrophoresed on denaturing agarose gels, transferred to membranes, and hybridized with strand-specific labeled riboprobes. Signals in the membrane were detected (A) and quantified (B) using a Storm 840 PhosphorImager. Removal of cyclobutane pyramidine dimers was calculated by comparing the amount of radioactivity in the T4 endonuclease V-treated versus mock-treated fragments and normalized by comparison with a loaded control plasmid (pGL3 control). The human telomerase reverse transcriptase-transfected fibroblasts were used for the assay at population doubling 65.

digested with SalI, an enzyme that makes one cut in the pCI- 68 was, as expected, significantly higher than that of the con- neo-hTERT plasmid but does not cut within the cellular hTERT trols, the luciferase activity of FT-1 at PD 92 was similar to that gene (43, 44). This enzyme should generate an 8.9-kb hTERT- of the control cells (Fig. 5C). Moreover, FT-1 and FT-3 cells at specific DNA fragment if the pCI-neo-hTERT plasmid exists as higher PDs (PD 84 and PD 86, respectively) showed similar an episome in cells and a single cellular hTERT band. As shown end-joining activity as the control cells, presumably due to loss in Fig. 5B, an 8.9-kb hTERT-specific band was identified in the of telomerase activity (Fig. 4, A and B). This important finding hTERT-transfected clones at PD 62 but was not seen in the showed that loss of telomerase activity caused the loss of parental NHOFs or in the vector-transfected control. This band enhanced DNA repair capacity in cloned NHOF of identical was absent in the hTERT-transfected clones at PD 85. At PD 62, genotype the FT-1 clone harbored more (22%) episomal exogenous hTERT gene than the FT-3 clone. An endogenous hTERT- specific band of approximately 23.1 kb was observed in all of DISCUSSION the tested cells. These results indicated that the exogenous Because the FT-1 and FT-3 NHOF clones transfected with hTERT cDNA existed as an episomal form in the hTERT- hTERT cDNA contained hTERT cDNA in an episomal form transfected fibroblast clones. The diminution and eventual arrest that was lost within 35 PDs after transfection, they provided us of telomerase activity in the hTERT-transfected clones was with critical unique features for investigating the role of hTERT/ correlated with loss of the exogenous hTERT plasmids. These telomerase on DNA repair. If the foreign DNA had integrated findings also revealed a quantitative correlation between the into host chromosomal DNA, its function or effect may not have number of surviving hTERT plasmids and telomerase activity as been precisely evaluated due to potential genetic alterations well as cellular replication capacity. caused by gene silencing or mutation (45, 46). Also, FT-1 and Using the host cell reactivation assay, we also compared FT-3 expressed hTERT and telomerase activity in a transient the repair capacity of FT-1 cells at PD 92 (after they had lost manner between PDs 41 and 84 but continued to divide expo- telomerase activity but were still dividing exponentially) with nentially for another 10 and 6 PDs, respectively. Therefore, we that of FT-1 cells at PD 68 (when they were showing telomerase were able to compare the effect of hTERT on DNA repair, not activity; Fig. 5C). Whereas the luciferase activity of FT-1 at PD only between transfected and parental (or control) cells, but also

during the pS189 plasmid DNA replication was not affected (Table 1). This is in agreement with the finding by Roques et al. (47) that the mutation frequency of microsatellite DNA in a shuttle vector was the same in parental and telomerase-immortalized human fibroblasts. Because of the gradual decrease of telomerase activity in hTERT-transfected NHOF during cellular replication, we determined the DNA repair capacity at their peak level of telomerase expression (PD 65) during exponential replication. The control cells were also tested during their exponential replication phase. Telomerase activity reduced the mutation frequency of the pS189 plasmid replicating in FT-1 and FT-3 cells treated with MNNG by 2-fold. This represented a 2-fold acceleration of the repair of damaged DNA, both cellular and plasmid, within 24 h after MNNG treatment. This enhancement of DNA repair activity by hTERT was confirmed by host cell reactivation of the firefly luciferase gene in the pGL3-Luc plasmid damaged in vitro by MNNG and transfected into hTERT/telomerase-expressing NHOF. Within 48 h after transfection of the plasmids damaged with 100 ng/ml MNNG, luciferase activity was twice as high in FT-1 and FT-3 as in the control cells. However, it should be noted that the normal repair mechanism could function without hTERT, albeit at a slower rate, because a signifi- cant level of DNA repair occurred in the absence of hTERT, especially at the low MNNG dose (50 ng/ml) in parental NHOF and Fneo cells. Hence, hTERT and telomerase activity enhanced DNA repair but is not required for DNA repair. It has also been reported that telomerase and ATM protect chromosome ends and double-stand breaks, thereby preventing chromosome rear- rangements (48, 49). We also demonstrated that hTERT/telom- Fig. 4 Enhancement of DNA end-joining activity by human telomer- erase activity accelerated in vitro DNA end-joining activity. ase reverse transcriptase/telomerase. The DNA end-joining assays were Because Ku proteins are key molecules in the DNA end-joining performed as described in “Materials and Methods.” A, the in vitro pathway and physically associate with hTERT (9), it appears end-joining assay was performed. Agarose gel electrophoresis of PCR- that hTERT may facilitate their recognition of broken DNA amplified rejoined plasmid DNA. FT-5 is a human telomerase reverse transcriptase-transfected clone that never expressed telomerase activity ends. hTERT binds to total genomic DNA independently of (data not shown). M, size marker. Negative control, linearized plasmids hTR, which is required to bind to telomeric DNA (21). It is well without incubation in cell extract. Positive control, nonlinearized plas- established that primary mammary epithelial cells that lack mids without incubation. B, quantification of in vitro end-joining activ- active telomerase develop chromosomal abnormalities and ity. Amplified DNA was quantified using Scion image (Scion Corp., Frederick, MD). The level of end-joining activity was normalized to the spontaneously become transformed when cultured in vitro (50, level of Fneo control. The assay was repeated three times. The error 51). Such cells are the progenitors of mammary carcinoma. P Ͻ 0.05 versus Fneo. C, the in vivo DNA The increased efficiency of DNA repair by hTERT was not ,ء .bars indicate ϮSD end-joining assay was performed. The relative precise DNA end-joining restricted to foreign episomal DNA, as demonstrated by the activity was calculated by comparing luciferase activity expressed in increased rate of NER of the endogenous cellular p53 gene. cells transfected with NarI-digested plasmid with that of the uncut plasmid. The results were obtained from three independent transfection Thus the effect of hTERT was not an artifact on transfected -P Ͻ 0.05 versus controls (N-2 and N-p). unintegrated plasmid DNA with a different association of his ,ء .experiments tones and other DNA-binding proteins normally associated with cellular chromosomal DNA. In hTERT/telomerase-expressing FT-1 cells, the NER rate was twice that of the control cells. In between the earlier stage of active hTERT and telomerase FT-3 cells expressing a lower level of hTERT/telomerase, the expression and the later stages, when hTERT and telomerase NER rate was faster than that in controls but slower than that in expression diminished and eventually ceased. This allowed us to FT-1, demonstrating a quantitative relationship between the accurately evaluate the effect of hTERT and telomerase activity level of hTERT and its accelerating effect on the DNA repair on DNA repair in a direct and quantitative manner. mechanism. The nontranscribed DNA strand being replicated The transient expression of telomerase activity in FT-1 and 3Ј35Ј via Okasaki fragments must wait for repair of the tran- FT-3 extended their in vitro life span by 20 and 10 PDs, scribed strand and its replication beyond the repaired site (52). respectively, as compared with control cells. The rate of cell Consequently, its repair rate was accelerated indirectly through division remained same as that of the control cells, suggesting acceleration of the complementary strand repair by hTERT, but that hTERT/telomerase has no effect on the cell cycle. Also, the the delay between repair of the two strands was unaffected by DNA proofreading (as detected by spontaneous mutagenicity) hTERT.

Fig. 5 Disappearance of enhanced DNA repair activities when the cells lost telomerase activity. A, in vitro replication of human telomerase reverse transcriptase (hTERT)- transfected normal human oral fibroblasts (NHOF). The cell population doubling (PD) level of parental, vector-transfected (Fneo), and hTERT-transfected NHOF (FT-1 and FT-3) was calculated as described in “Mate- rials and Methods.” Arrow indicates the PD at which empty vector or hTERT cDNA was transfected into NHOF. B, episomal status of hTERT cDNA. Southern blot hybridization of SalI-digested cellular DNA to 32P-labeled, endogenous hTERT gene ,ء .hTERT cDNA exogenous hTERT plasmid ,ءء .fragment cDNA. C, host cell reactivation assay of luciferase activity in the FT-1 clone at PD 68 (expressing telomerase activity) and PD 92 P Ͻ ,ء .(after loss of telomerase activity) 0.05 versus FT-1 (PD 92).

To eliminate the unlikely possibility that the enhanced ulate that hTERT facilitates DNA repair by recruiting the initi- DNA repair activity of the hTERT-expressing clones (FT-1 and ation factors to the DNA damage sites. However, unlike the FT-3) was due to some sort of selectivity, we transfected another report of Sharma et al. (21), our in vitro and in vivo end-joining strain of NHOF with the pCI-neo-hTERT plasmid. We observed assays established a direct involvement of hTERT in double- similar results from two independent hTERT transfection stud- strand break repair. The different experimental approaches used ies. This indicated that hTERT expression is associated with could be responsible for this discrepancy. Because our in vitro increased DNA repair efficiency for different types of DNA assay was PCR based, it was more sensitive than that used by damage induced in NHOF. Sharma et al. (21). Moreover, using our in vivo assay, we could The increased efficiency of DNA repair was detected for selectively measure accurate DNA end-joining activity in cells. different types of DNA damage that presumably required dif- The increased intracellular level of ATP induced by hTERT ferent repair mechanisms. The Sharma et al. (21) report added reported by Sharma et al. (21) as responsible for faster DNA repair of ionizing radiation and DNA adducts to our data. repair kinetics is another possible mechanism by which hTERT Therefore, the role of hTERT in DNA repair did not appear to accelerates DNA repair. However, the two possibilities are not be restricted to a specific repair mechanism but to general factors involved in all forms of DNA repair. Some protein mutually exclusive. factors involved in DNA repair, DNA replication, or telomere maintenance, e.g., RAD-50, MRE-11, NBS, Ku, TRF-1, and ACKNOWLEDGMENTS TRF-2, form physical complexes with each other and with We thank Dr. R. A. Weinberg for the pCI-neo-hTERT plasmid and hTERT (2, 9, 13–15, 53, 54). Therefore, it is not surprising that Dr. E. J. Shillitoe (SUNY-Syracuse) for the shuttle vector pS189 and E. hTERT can accelerate the DNA repair process in a concentra- coli strain MBM7070. tion-dependent manner. DNA damage itself does not induce hTERT expression.3 Judging from the ability of hTERT to form complexes with protein factors that bind to DNA ends and the REFERENCES kinetics of DNA repair in presence of excess hTERT, we spec- 1. Greider CW. Telomere length regulation. Annu Rev Biochem 1996; 65:337–65. 2. Mergny JL, Riou JF, Mailliet P, Teulade-Fichou MP, Gilson E. Natural and pharmacological regulation of telomerase. Nucleic Acids 3 Unpublished data. Res 2002;30:839–65.

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Ki-Hyuk Shin, Mo K. Kang, Erica Dicterow, et al.

Clin Cancer Res 2004;10:2551-2560.

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