Vol. 10, 2551–2560, April 1, 2004 Clinical Cancer Research 2551 Introduction of Human Telomerase Reverse Transcriptase to Normal Human Fibroblasts Enhances DNA Repair Capacity 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 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2004 American Association for Cancer Research. 2552 Telomerase Activity Enhances DNA Repair 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.