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Experimental Therapeutics, Preclinical Pharmacology |
1 School of Dentistry and 2 Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: To study the effect of hTERT/telomerase on DNA repair, we examined the mutation frequency rate, host cell reactivation rate, nucleotide excision repair capacity, and DNA end-joining activity of NHOF and NHOF capable of expressing hTERT/telomerase (NHOF-T). NHOF-T was obtained by transfecting NHOF with hTERT plasmid.
Results: Compared with parental NHOF and NHOF transfected with empty vector (NHOF-EV), we found that (a) the N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation frequency of an exogenous shuttle vector was reduced in NHOF-T, (b) the host cell reactivation rate of N-methyl-N'-nitro-N-nitrosoguanidine-damaged plasmids was significantly faster in NHOF-T; (c) the nucleotide excision repair of UV-damaged DNA in NHOF-T was faster, and (d) the DNA end-joining capacity in NHOF-T was enhanced. We also found that the above enhanced DNA repair activities in NHOF-T disappeared when the cells lost the capacity to express hTERT/telomerase.
Conclusions: These results indicated that hTERT/telomerase enhances DNA repair activities in NHOF. We hypothesize that hTERT/telomerase accelerates DNA repair by recruiting DNA repair proteins to the damaged DNA sites.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The involvement of DNA repair proteins in telomere maintenance has been well documented (5, 6, 7, 8) . In eukaryotic cells, nonhomologous end-joining requires a DNA ligase and the DNA-activated protein kinase, which is recruited to the DNA ends by the DNA-binding protein Ku. Ku binds to hTERT without the need for telomeric DNA or hTR (9) , binds the telomere repeat-binding proteins TRF1 (10) and TRF2 (11) , and is thought to regulate the access of telomerase to telomere DNA ends (12 , 13) . The RAD50, MRE11, and NBS1 proteins, which are involved in DNA repair, are also active in telomere elongation via protein kinase ATM and associate with TRF1 and TRF2 (13, 14, 15, 16, 17) . Moreover, recent observations indicate that telomerase also associates with proteins known to participate in DNA replication (18) and in DNA repair (9) . Also, DNA repair is impaired in mice with telomere dysfunction (19 , 20) . Because proteins involved in DNA replication are required for repair of damaged DNA, there exists a possibility that telomerase is also involved in DNA repair. A recent report showed that ectopic expression of hTERT accelerated the repair of DNA double-strand breaks induced by ionizing radiation and of DNA adducts produced by cisplatin (21) .
To investigate the putative role of hTERT in general DNA repair, two independent strains of normal human oral fibroblasts (NHOF) were transfected with a plasmid capable of expressing hTERT. NHOF do not express the hTERT gene, which is silenced by hypermethylation (22) . They express hTR, which is ubiquitously present in normal cells. Three NHOF clones that expressed hTERT and telomerase activity were established. Two of the three clones transiently expressed telomerase activity because the nonintegrated plasmids were lost after approximately 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 (NHOF-EV). After the hTERT-transfected cells ceased to express hTERT and telomerase activity, they lost the capacity to enhance DNA repair. The hTERT-induced enhancement of DNA repair was demonstrated in four different ways. First, in NHOF expressing telomerase activity treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the mutation frequency of the replicating pS189 shuttle vector was decreased. Second, NHOF expressing telomerase activity had a higher repair level of exogenous DNA damaged in vitro by MNNG. Third, NHOF expressing telomerase activity showed a faster rate of nucleotide excision repair (NER) in both strands of an endogenous cellular gene. Fourth, NHOF expressing telomerase activity had a higher rate of DNA end-joining activity. The parental primary NHOF and five hTERT-negative clones served as controls.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection and Cloning.
A human TERT expression plasmid (pCI-neo-hTERT) was provided by Dr. Robert A. Weinberg (Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology). Control plasmid (pCI-neo) was obtained from Promega (Madison, WI). After 41 PDs, approximately 2 x 105 exponentially replicating NHOFs per 60-mm culture dish were transfected with pCI-neo-hTERT or pCI-neo by using LipofectAMINE 2000 reagent (Invitrogen). For each 60-mm dish, a mixture of pCI-neo-hTERT or pCI-neo (5 µg/50 µl) and the LipofectAMINE reagent (35 µg/50 µl) was added to the culture medium dropwise as uniformly as possible with gentle swirling. The cells were incubated for 7 h at 37°C. The medium was then replaced with fresh culture medium, and the cultures were incubated for an additional 24 h. To select cells transfected with the pCI-neo-hTERT or pCI-neo, the cells were incubated in culture medium containing 200 µg/ml G418 (Invitrogen). Then, G418-resistant clones were isolated by ring-cloning. The G418-resistant clones transfected with pCI-neo-hTERT or pCI-neo were selected and subcultured.
Nucleic Acid Isolation.
High molecular weight cellular DNA was extracted from the cells with phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol precipitation (24)
.
Analysis of Telomerase Activity.
Cellular extracts were prepared by using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (lysis buffer) provided from the TRAP-eze Telomerase Detection Kit (Intergen Corp., Norcross, GA) as recommended by the manufacturer. Telomerase activity was determined using the TRAP-eze Telomerase Detection Kit as described previously (23)
. Each telomeric repeat amplification protocol reaction contained cellular extract equivalent to 1 µg protein. The PCR products were electrophoresed in 12.5% nondenaturing polyacrylamide gels, and the radioactive signals were detected by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Southern Blot Analysis.
For genomic Southern blot analysis, 10 µg of DNA were digested with SalI restriction enzyme. The fragmented DNA was then separated by electrophoresis, transferred to nitrocellulose filter, and hybridized to 32P-labeled full-length hTERT cDNA. The hTERT cDNA was labeled with [32P]dCTP (ICN Radiochemicals, Irvine, CA) by Prime-It RmT Random Primer Labeling Kit (Stratagene). The radioactive signals were detected by PhosphorImager and quantitatively analyzed by ImageQuant software (Molecular Dynamics Inc., Sunnyvale, CA).
Analysis of Mutation Frequency in pS189 Shuttle Vector.
The shuttle vector pS189 and host Escherichia coli strain MBM7070 obtained from Dr. E. J. Shillitoe (State University of New York, Syracuse, NY) were described elsewhere (25
, 26)
. The pS189 vector can replicate in a number of different cell types, including lymphoblasts (24)
, cells from xeroderma complementation groups (27)
, normal oral human keratinocytes, and oral squamous cell carcinoma cells (26
, 28)
. Eighty percent confluent cells were transiently transfected with pS189 plasmids using LipofectAMINE 2000 reagent (Invitrogen) as described previously. Twenty-four h after transfection, the cells were exposed to 1.5 µg/ml MNNG for 2 h and cultured in fresh medium not containing the chemical for an additional 24 h. At the end of the incubation, the plasmids were recovered from the cells by the alkaline lysis method as described elsewhere (29)
. Plasmid DNA was digested with the enzyme DpnI (Boehringer Mannheim Biochemicals, Indianapolis, IN) to cleave DNA from plasmids that had not replicated in NHOF (30)
. Recovered plasmid DNA that had replicated in cells were transfected into E. coli MBM7070 by the heat shock method. Transformed bacteria were plated on Luria-Bertani agar plates containing ampicillin (50 µg/ml), 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside, and isopropyl-1-thio-ß-D-galactopyranoside. Bacterial colonies containing plasmids with the mutant or wild-type suppressor tRNA gene (supF) were identified by color (cells containing wild-type plasmids are blue, whereas cells with mutant plasmids are light blue or white). The mutagenic frequency was determined as the percentage of white and light blue colonies in the total colonies [mutagenesis frequency (%) = number of white and light blue colonies ÷ number of total colonies].
Host Cell Reactivation Assay.
The pGL3-Luc plasmid (Promega), in which expression of the firefly luciferase gene is controlled by the cytomegalovirus (CMV) promoter, was used to determine the capacity of cells for repairing damaged DNA. The pRL-CMV plasmid, in which the Renilla luciferase gene is driven by the CMV promoter, was used as an internal control for transfection efficiency. To create in vitro damaged DNA, the pGL3-Luc plasmid was exposed to 50 or 100 ng/ml MNNG for 30 min in Tris-EDTA buffer and purified with Wizard DNA Clean-Up System (Promega). Approximately 5 x 104 cells/well were plated in a 24-well culture dish and cultured for 24 h. The cells were then transiently transfected with 1 µg damaged pGL3-Luc plasmid/well and 0.1 µg pRL CMV plasmid/well using the LipofectAMINE reagent following the manufacturer’s instructions. The pRL-CMV plasmid was used to normalize for total DNA transfected. After 4 h of transfection, the transfection medium was replaced with regular culture medium. Cells were collected 48 h after transfection, and cell lysates were prepared according to the Promega’s instruction manual. Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega) and a luminometer (Promega). The Renilla luciferase activity was used to normalize for transfection efficiency.
NER Assay.
Strand-specific riboprobes for the p53 EcoRI fragment detection were prepared by PCR amplification of a human genomic DNA fragment between nucleotides 1750 and 2138 of exon II of the p53 gene. This fragment of 388 nucleotides was ligated into the pGEM-T plasmid (Promega) and sequenced to confirm the absence of mutations. Strand-specific 32P-labeled riboprobes were generated using the T7 or SP6 transcription promoters in the pGEM-T/p53 vector as described previously (31)
. Strand-specific removal of UV-induced cyclobutane pyramidine dimers (CPDs) was analyzed from a 16-kb EcoRI fragment of the active p53 gene using single-stranded labeled riboprobes specific for p53 (32
, 33)
. Cells cultured to confluence were UV-irradiated with 2.5 J/m2 (254 nm). Genomic DNA was extracted at 0, 8, and 24 h after UV-irradiation using phenol/chloroform/isoamyl alcohol (25:24:1) and ethanol precipitation (24)
. The extracted DNA was 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 to Hybond-N nylon membranes (Amersham Life Sciences, Arlington Heights, IL). After hybridization with strand-specific riboprobes, signals in the membrane were detected using a Storm 840 phosphorimager and quantified with ImageQuant software, version 1.2 (Molecular Dynamics). Repair of CPDs was calculated by comparing the amount of radioactivity in the T4V-treated versus mock-treated fragments and normalized with a loaded control plasmid (pGL3 control plasmid).
In Vitro DNA End-Joining Assay.
Cells were collected 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, 150 mM NaCl, 10 mM Tris (pH 7.4), 1 mM EDTA, 1 mM EGTA (pH 8.0), 0.2 mM sodium orthovanadate, and protease inhibitor mixture (Boehringer Mannheim)]. The cell lysates were centrifuged at 8000 x g for 10 min at 4°C. EcoRI linearized pCR2.1-TOPO plasmid (Invitrogen) was incubated with total cell extracts for 2 h at 37°C in 20 µl of reaction mixture containing 1 µl of linearized plasmid (10 ng), 2 µl of cell extract (10 µg), 4 µl of 50% polyethylene glycol, and 2 µl of 10x ligase buffer [300 mM Tris-HCl (pH 7.8), 100 mM KC1, 100 mM DTT, and 10 mM ATP]. To amplify rejoined DNA, PCR reaction was performed with 3 µl of end-joining reaction using M13 reverse primer (5'-CAGGAAACAGCTATGAC- 3') and M13 forward primer (5'-GTAAAA CGACGGCCAG-3'). The PCR condition consisted of 35 cycles at 95°C for 30 s, 60°C for 30 s, and 70°C for 30 s. PCR products were separated in 2% agarose gel electrophoresis in Tris-borate EDTA buffer and visualized by staining with ethidium bromide. Amplification of rejoined DNA was evident as a 186-bp band.
In Vivo DNA End-Joining Assay.
The pGL3 plasmid (Promega), in which expression of the luciferase gene is controlled by the CMV promoter, was used to evaluate correct nonhomologous end-joining activity that precisely rejoins broken DNA ends in vivo. The pGL3 plasmid was completely linearized by restriction endonuclease NarI (New England Biolabs), which cleaves within the luciferase coding region as confirmed by agarose gel electrophoresis. The linearized DNA was subjected to phenol/chloroform extraction and ethanol precipitation and dissolved in sterilized water.
Before transfection, a 6-well plate was inoculated with approximately 5 x 104 cells/well and cultured for 24 h. The cells were then transiently transfected with 1 µg linearized pGL3 plasmid/well or 1 µg intact pGL3 plasmid/well using the LipofectAMINE reagent (Invitrogen) following the manufacturer’s instructions. After 7 h of transfection, the transfection medium was replaced with regular culture medium. Cells were collected 48 h after transfection, and cell lysates were prepared according to the Promega instruction manual. Luciferase activity was measured using the Luciferase Reporter Assay System (Promega) and a luminometer (Promega). The reporter plasmid was digested to completion with NarI within the luciferase coding region, and only precise DNA end-joining activity should restore the luciferase activity. The precise nonhomologous end-joining activity was calculated from the luciferase activity of linearized pGL3 plasmids compared with that of the uncut plasmids. Each experiment was repeated three times.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The telomerase-positive NHOF clones were serially subcultured to determine the effect of telomerase on replication and senescence of NHOF. Also, telomerase activity was tested again in FT-1 at PDs 62, 74, and 84 and in FT-3 at PDs 62, 76, and 86 (Fig. 1, A and B)
. At PDs 62 and 74, FT-1 showed telomerase activity, which was decreased by 5.5-fold in cells at PD 74. A similar pattern of reduced telomerase activity was noted in FT-3 at higher PDs. Telomerase activity was not detected in FT-1 and FT-3 at PD 84 and PD 86, respectively, although these clones continued to replicate exponentially (Fig. 1, A and B)
. FT-3 expressed approximately four times less telomerase activity than FT-1 at all times tested. Thus, telomerase activity was only transiently expressed in the hTERT-transfected NHOF clones.
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. The parental NHOF and three clones transfected with the empty vector (N-1, N-2, and N-p) did not display telomerase activity.
hTERT/Telomerase Decreases the Mutation Frequency of a Shuttle Vector in NHOF Treated with a DNA-Damaging 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 expression of telomerase activity at PD 74 and a total loss of activity at PD 84 (Fig. 1)
, we used cells at PD 65. The spontaneous mutation frequency (1/23,049) of the pS189 plasmid in the clones FT-1 and FT-3 was similar to that (1/28,131) in parental NHOF and in Fneo analyzed at PD 60 and PD 62, respectively during the exponential replication phase (Table 1)
. After the cells were treated with MNNG, the mutation frequency of the shuttle vector was significantly increased in all of the tested cells. However, the magnitude of the increase was almost two times lower in FT-1 and FT-3 cells than that in the parental and Fneo cells (Table 1)
. These data indicated that the expression of hTERT/telomerase activity either prevented the mutation of the plasmids or increased the repair of DNA damaged by MNNG.
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The pGL3-Luc luciferase reporter plasmids were treated with 0, 50, or 100 ng/ml MNNG. The damaged plasmids were transiently transfected into parental NHOF (PD 60), Fneo (PD 62), FT-1 (PD 65), and FT-3 (PD 65). The luciferase activity was monitored in all tested cells. The hTERT-expressing fibroblasts demonstrated a significantly higher level of luciferase activity compared with the controls. At the higher (100 ng/ml) concentration of MNNG, the level was twice as high as in controls. The luciferase activity of undamaged plasmids was similar in the controls and the hTERT-transfected cells (Fig. 2A)
.
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hTERT/Telomerase Accelerates the NER Process of Cellular Gene.
In mammalian cells, a variety of DNA lesions such as UV-induced CPDs are repaired by the NER pathway (38)
. The NER pathway is divided into transcription coupled repair, which is restricted to the transcribed strand of transcriptionally active genes, and general genome repair, which acts on DNA lesions within the entire genome (38
, 39)
.
The effect of hTERT/telomerase on NER of the endogenous p53 gene was measured by the rate of removal of UV-induced CPDs from the individual strands of the gene sequence (40)
. This assay involves both transcribing strand-specific NER and general NER. The parental, vector-transfected, and hTERT/telomerase-expressing fibroblasts at PD 65 were irradiated with 2.5 J/m2, and cellular DNA was isolated 0, 8, and 24 h after UV-irradiation. The extracted DNA was treated with T4V, which cleaves DNA containing UV-induced CPDs. The strand-specific removal of CPDs from a 16-kb EcoRI fragment of the 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% after 24 h. The slower rate of NER repair in FT-3 than in FT-1 appears to reflect the lower expression of telomerase activity resulting from fewer active hTERT plasmids in FT-3. The delay in repairing the nontranscribed strand was not affected by telomerase activity. The NER assay was not performed with the T-11 clone.
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. 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 also examined in this experiment. One of the 13 hTERT-transfected clones, FT-5, which failed to demonstrate telomerase activity, showed end-joining activity similar to that of the control (Fig. 4, A and B)
.
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. These data confirmed the results obtained from the hTERT-transfected NHOF-1 strain and indicated that hTERT/telomerase expression was associated with the enhancement of in vitro and in vivo DNA end-joining activity in NHOF.
Enhanced DNA Repair Activity Disappears When the Cells Lost Telomerase Activity.
We also compared the replication capacity of the hTERT-transfected NHOF-1 clones (FT-1 and FT-3) with that of the parental cells and the empty vector-transfected cells (Fig. 5A)
. The parental cells and the vector-transfected control ceased dividing at PD 82 and PD 80, respectively, whereas the hTERT-transfected clones, FT-1 and FT-3 replicated for an additional 20 and 10 doublings, respectively. The T-11 clone is replicating at present, and the cells have not yet reached senescence.
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. The cellular DNA was digested with SalI, an enzyme that makes one cut in the pCI-neo-hTERT plasmid but does not cut within the cellular hTERT gene (43
, 44)
. This enzyme should generate an 8.9-kb hTERT-specific DNA fragment if the pCI-neo-hTERT plasmid exists as an episome in cells and a single cellular hTERT band. As shown in Fig. 5B
, an 8.9-kb hTERT-specific band was identified in the hTERT-transfected clones at PD 62 but was not seen in the parental NHOFs or in the vector-transfected control. This band was absent in the hTERT-transfected clones at PD 85. At PD 62, 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 the tested cells. These results indicated that the exogenous hTERT cDNA existed as an episomal form in the hTERT-transfected fibroblast clones. The diminution and eventual arrest of telomerase activity in the hTERT-transfected clones was correlated with loss of the exogenous hTERT plasmids. These findings also revealed a quantitative correlation between the number of surviving hTERT plasmids and telomerase activity as well as cellular replication capacity.
Using the host cell reactivation assay, we also compared the repair capacity of FT-1 cells at PD 92 (after they had lost telomerase activity but were still dividing exponentially) with that of FT-1 cells at PD 68 (when they were showing telomerase activity; Fig. 5C
). Whereas the luciferase activity of FT-1 at PD 68 was, as expected, significantly higher than that of the controls, the luciferase activity of FT-1 at PD 92 was similar to that of the control cells (Fig. 5C)
. Moreover, FT-1 and FT-3 cells at higher PDs (PD 84 and PD 86, respectively) showed similar end-joining activity as the control cells, presumably due to loss of telomerase activity (Fig. 4, A and B)
. This important finding showed that loss of telomerase activity caused the loss of enhanced DNA repair capacity in cloned NHOF of identical genotype
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The transient expression of telomerase activity in FT-1 and FT-3 extended their in vitro life span by 20 and 10 PDs, respectively, as compared with control cells. The rate of cell division remained same as that of the control cells, suggesting that hTERT/telomerase has no effect on the cell cycle. Also, the DNA proofreading (as detected by spontaneous mutagenicity) 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 significant 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 rearrangements (48 , 49) . We also demonstrated that hTERT/telomerase activity accelerated in vitro DNA end-joining activity. Because Ku proteins are key molecules in the DNA end-joining pathway and physically associate with hTERT (9) , it appears that hTERT may facilitate their recognition of broken DNA ends. hTERT binds to total genomic DNA independently of hTR, which is required to bind to telomeric DNA (21) . It is well established that primary mammary epithelial cells that lack active telomerase develop chromosomal abnormalities and spontaneously become transformed when cultured in vitro (50 , 51) . Such cells are the progenitors of mammary carcinoma.
The increased efficiency of DNA repair by hTERT was not restricted to foreign episomal DNA, as demonstrated by the increased rate of NER of the endogenous cellular p53 gene. Thus the effect of hTERT was not an artifact on transfected unintegrated plasmid DNA with a different association of histones 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 FT-3 cells expressing a lower level of hTERT/telomerase, the NER rate was faster than that in controls but slower than that in FT-1, demonstrating a quantitative relationship between the level of hTERT and its accelerating effect on the DNA repair mechanism. The nontranscribed DNA strand being replicated 3'
5' via Okasaki fragments must wait for repair of the transcribed strand and its replication beyond the repaired site (52)
. Consequently, its repair rate was accelerated indirectly through acceleration of the complementary strand repair by hTERT, but the delay between repair of the two strands was unaffected by hTERT.
To eliminate the unlikely possibility that the enhanced DNA repair activity of the hTERT-expressing clones (FT-1 and FT-3) was due to some sort of selectivity, we transfected another strain of NHOF with the pCI-neo-hTERT plasmid. We observed similar results from two independent hTERT transfection studies. This indicated that hTERT expression is associated with increased DNA repair efficiency for different types of DNA damage induced in NHOF.
The increased efficiency of DNA repair was detected for different types of DNA damage that presumably required different repair mechanisms. The Sharma et al. (21) report added repair of ionizing radiation and DNA adducts to our data. Therefore, the role of hTERT in DNA repair did not appear to be restricted to a specific repair mechanism but to general factors involved in all forms of DNA repair. Some protein factors involved in DNA repair, DNA replication, or telomere maintenance, e.g., RAD-50, MRE-11, NBS, Ku, TRF-1, and TRF-2, form physical complexes with each other and with hTERT (2 , 9 , 13, 14, 15 , 53 , 54) . Therefore, it is not surprising that hTERT can accelerate the DNA repair process in a concentration-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 kinetics of DNA repair in presence of excess hTERT, we speculate that hTERT facilitates DNA repair by recruiting the initiation factors to the DNA damage sites. However, unlike the report of Sharma et al. (21) , our in vitro and in vivo end-joining assays established a direct involvement of hTERT in double-strand break repair. The different experimental approaches used could be responsible for this discrepancy. Because our in vitro assay was PCR based, it was more sensitive than that used by Sharma et al. (21) . Moreover, using our in vivo assay, we could selectively measure accurate DNA end-joining activity in cells. The increased intracellular level of ATP induced by hTERT reported by Sharma et al. (21) as responsible for faster DNA repair kinetics is another possible mechanism by which hTERT accelerates DNA repair. However, the two possibilities are not mutually exclusive.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: No-Hee Park, University of California Los Angeles School of Dentistry, CHS 53-038, 10833 Le Conte Avenue, Los Angeles, CA 90095-1668. Phone: (310) 206-6063; Fax: (310) 794-7734; E-mail: npark{at}dent.ucla.edu
Received 5/ 1/03; revised 12/29/03; accepted 12/31/03.
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, luciferase activity of undamaged plasmids; 
, luciferase activity of plasmids damaged by 50 ng/ml MNNG; 
, luciferase activity of plasmids damaged by 100 ng/ml MNNG. The error bars indicate ±SD from three different assays. *, P < 0.05 versus controls. B, host cell reactivation assay of luciferase activity in empty vector-transfected clones (N-1, N-2, and N-p) and in hTERT-transfected fibroblast clone expressing telomerase activity (T-11). *, P < 0.05 versus controls (N-1, N-2, and N-p).
-32P-labeled riboprobes. DNA was extracted from the cells 8 and 24 h after UV-irradiation (hpUV), digested with EcoRI, and treated (+) or mock treated (–) 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.
