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Normal human oral keratinocytes demonstrate abnormal DNA end joining activity during replicative senescence
Mechanisms of Ageing and Development 126 (2005) 475–479 www.elsevier.com/locate/mechagedev
Normal human oral keratinocytes demonstrate abnormal DNA end joining activity during replicative senescence Mo K. Kang1, Ki-Hyuk Shin1, Felix K. Yip, No-Hee Park* School of Dentistry, University of California, Los Angeles, UCLA, CHS 53-038, 10833 Le Conte Avenue, Los Angeles, CA 90095-1668, USA Received 9 July 2004; received in revised form 30 September 2004; accepted 13 October 2004 Available online 9 December 2004
Abstract Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of cellular genetic integrity. DSBs are repaired by cellular end joining activity, which could proceed with varying degrees of accuracy. Abnormal end joining may lead to an accumulation of mutations and contribute to genetic instability and cellular aging. In the present study, we compared the efficiency and accuracy of end joining activities in exponentially replicating and senescing normal human oral keratinocytes (NHOK). We developed an in vitro end joining assay utilizing a plasmid linearized with a unique EcoR I or EcoR V restriction site. The efficiency of end joining was determined by PCR with primers that could amplify the fragment containing the end joining site. The accuracy of end joining was assessed by determining whether the original EcoR I site was restored after end joining. Both replicating and senescing cultures of NHOK yielded a similar level of end joining efficiency, which was noted by the similar intensity of PCR amplification. However, the frequency of end joining errors was significantly elevated in NHOK during replicative senescence. Senescing NHOK could thus accumulate abnormal end joining products, which might contribute to cellular aging and cancer. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Keratinocytes; Senescence; End joining; Cancer
1. Introduction Accumulation of DSBs or abnormal DSB repair products may contribute to genetic instability and aging process. A recent study demonstrated that senescing human fibroblasts and aging mice cells accumulate unrepairable DSBs (Sedelnikova et al., 2004), suggesting that DSBs and resulting genetic instability have a causal role in cellular and organismic aging. Also, human diploid fibroblasts (HDFs) showed decreased efficiency and increased frequency of errors in end joining activity during replicative senescence further supporting the important role of DSB and abnormal DSB repair in aging (Seluanov et al., 2004). Recently, we developed a PCR-based assay for end joining activity in vitro, using whole cell lysates and pCR2.1
plasmid DNA linearized with unique EcoR I or EcoR V restriction enzyme. We employed this method to quantitatively compare the efficiency and the error frequency in end joining activity of normal human oral keratinocytes (NHOK) during serial subculture. Exponentially replicating and senescing NHOK demonstrated a similar level of end joining efficiency, while the frequency of end joining errors was found to be significantly elevated in NHOK during senescence. Thus, loss of end joining fidelity and consequent accumulation of abnormal DSB repair products may contribute to the onset of senescence in these cells.
2. Materials and methods 2.1. Cells and culture conditions
* Corresponding author. Tel.: +1 310 206 6063; fax: +1 310 794 7734. E-mail address: [email protected] (N.-H. Park). 1 Contributed equally to this work.
Primary NHOK cultures from three donors (00-4, 00-5 and 00-6) were prepared from separated epithelial tissue and
0047-6374/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2004.10.002
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M.K. Kang et al. / Mechanisms of Ageing and Development 126 (2005) 475–479
serially subcultured in keratinocyte growth medium (KGM, Cambrex Corp., East Rutherford, NJ, USA) containing 0.15 mM Ca++ as described previously (Kang et al., 2000). For each passage, 105 cells were plated in 100 mm culture dish and transferred when the culture reached 70% confluence. Another NHOK culture (01-7) was maintained in a feeder cell system with NIH 3T3 fibroblasts in serumcontaining medium (Kang et al., 2004). Two million mitomycin C-treated 3T3 cells and 3 105 NHOK were plated per 100 mm dish and the cells were allowed to proliferate to 70% confluence. At the time of transfer, the 3T3 cells were removed with 0.02% EDTA in 1 phosphatebuffered saline, and NHOK were trypsinized and replated on fresh feeder cells. Under these conditions, NHOK exhibited the typical first-order replication kinetics, albeit the replication potential was influenced by the culture conditions (Kang et al., 2004).
2.2. In vitro DNA end joining assay Cells were collected and washed three times in ice-cold phosphate-buffered saline (PBS). The cells were lysed by incubation for 30 min at 4 8C in lysis buffer (1% Triton X100; 150 mM NaCl; 10 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA, pH 8.0; 0.2 mM sodium ortho-vanadate). The cell lysates were cleared by centrifugation at 8000 g for 10 min at 4 8C. The pCR2.1-TOPO plasmid (Invitrogen, Carlsbad, CA, USA) was linearized by restriction with EcoR I to generate cohesiveness. The complete digestion was confirmed by agarose gel electrophoresis. The linearized DNA was subjected to phenol/chloroform extraction, ethanol-precipitated, and dissolved in sterilized water. The in vitro DNA end joining reactions (20 ml) were performed with 5 mg total cellular extract and 10 ng linearized plasmid in the presence of 4 ml of 50% polyethyleneglycol (PEG; Sigma) and 2 ml of 10 ligase buffer (300 mM Tris–HCl, pH 7.8; 100 mM KC1; 100 mM DTT and 10 mM ATP) at 37 8C for 2 h. After the end joining reaction, PCR reaction was performed with 3 ml end joining reaction using M13 reverse primer (50 CAGGAAACAGCTATGAC-30 ) and M13 forward primer (50 -GTAAAACGACGGCCAG-30 ) to amplify rejoined DNA. The PCR amplification was allowed for 30 cycles at 95 8C for 30 s, 60 8C for 30 s, and 70 8C for 30 s. PCR products were separated in 2% agarose gel and visualized by staining with ethidium bromide. Accurate end joining of the plasmid linearized with EcoR I would yield 186 bp PCR fragments. The above PCR condition allowed for exponential amplification of the DNA templates. Blunt ended DNA was prepared by complete digestion of the pCR2.1/GPR1 plasmid with EcoR V (New England Biolabs). This plasmid contained an additional 371 bp of partial GPR1 fragment cloned into a cloning site (overhanging 30 deoxythymidine residues) outside the unique EcoR V restriction site within the pCR2.1 backbone.
Accurate end joining of the plasmid linearized with EcoR V was amplified as a band of 573 bp. The efficiency of end joining was determined by noting the intensity of the 186 or 573 bp PCR fragment and was quantitated using Scion image analysis software (Scion Corp., Frederek, MD, USA). The accuracy of end joining was assessed by cloning the PCR fragment into pcDNA3.1/ V5-His TOPO plasmid (Invitrogen). The resulting ligation products were introduced into Eschericia coli strain TOP10 (Invitrogen). Subsequently, single colony PCR was performed using the M13 primer set. The PCR product was digested with EcoR I or EcoR V and electrophoresed in 2% agarose gel to compare the restriction profiles. The PCR products, which were resistant to the EcoR I or EcoR V digestion, were considered as abnormal end joining with sequence alteration. The PCR products sensitive to the enzyme digestion represented the precise end joining activity. The frequency of abnormal end joining was determined by noting the number of E. coli colonies, which gave rise to the PCR product resistant to the enzyme digestion. 2.3. DNA sequencing analysis To determine the exact sequence alterations in abnormal end joining, the PCR fragments resistant to either EcoR I or EcoR V digestion were sequenced directly with a Taq dideoxy terminator cycle sequencing kit on an ABI 377 automatic DNA sequencer (Perkin–Elmer; UCLA Sequencing Core Facility).
3. Results 3.1. Senescing NHOK demonstrated increased frequency of abnormal end joining activity We developed a PCR-based in vitro end joining assay for quantitative determination of both precise and abnormal end joining activities (Shin et al., manuscript submitted). pCR2.1 plasmid was linearized at the unique EcoR I restriction site to create cohesive DNA ends and incubated with whole cell lysates. Plasmid end joining was detected by PCR amplification of the plasmid sequences containing the EcoR I site, yielding 186 bp fragments. To demonstrate the efficiency of (or lack thereof) end joining simply by DNA end annealing event, we also performed the in vitro end joining assay in the absence of cell lysate. We found that there was no amplification of the substrate DNA without exposure to the cell lysate (Shin et al., manuscript submitted). This finding indicates complete digestion of the plasmid DNA and lack of false amplification resulting from sticky end annealing. We also performed the in vitro DNA end joining assay and varied (1) the amount of cell lysate, (2) number of PCR amplification cycles, or (3) amount of linearized DNA substrate. We found that the level
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477
Fig. 1. Senescent NHOK demonstrate abnormal end joining activity in vitro. Whole cell extracts of NHOK at PD 14 and PD 16 were incubated with pCR2.1 plasmid DNA linearized with EcoR I. The level of end joining was determined by PCR amplification of regions containing the restriction site (A). Restoration of the original EcoR I restriction sequence was determined by isolating the PCR fragments from (A) and performing single-colony PCR followed by EcoR I digestion (B). The doublets indicate EcoR I-sensitive fragment, while the high molecular weight single band was resistant to the enzyme digestion.
of DNA substrate amplification using the PCR conditions described in this study is quantitatively correlated to the above variables (Shin et al., manuscript submitted). Therefore, efficiency of cellular end joining activity would be correlated to the intensity of PCR amplification, while the accuracy of end joining could be assessed by the molecular size of the PCR products and restoration of the EcoR I restriction sequence. To determine the effects of replicative senescence on the efficiency and accuracy of end joining activity in NHOK, the in vitro end joining assay was performed with exponentially replicating and senescing culture of NHOK (Fig. 1A). Both NHOK cultures at PD 14 and PD 26 demonstrated a similar level of end joining efficiency and the reaction yielded the expected 186 bp fragment. Also, end joining in NHOK proceeded without gross sequence alteration of the end joining site regardless of the cellular replication status. The 186 bp PCR fragment presumably contained a variety of end joining reaction products, which may possess errors not detectable by agarose gel electrophoresis. Therefore, the accuracy of end joining was also determined by restoration of the EcoR I site in the individual PCR fragments. The 186 bp fragments obtained from end joining activity of exponentially replicating and senescing NHOK were excised from the agarose gel and cloned into pcDNA3.1/V5-His TOPO plasmid. Competent E. coli was transformed with the resulting mixture of constructs and single colony PCR amplification was performed using the M13 primers. The PCR products were digested with EcoR I and electrophoresed in 2% agarose (Fig. 1B). The colonies, which gave rise to EcoR I-resistant PCR fragments were scored as representing abnormal end joining activity. Using this method, we have tested the accuracy of end joining activity in three different strains of NHOK (03-1, 02-1, and 02-7) at the exponentially replicating and senescing phases
of replication (Table 1). We found that the vast majority of the colonies yielded EcoR I-sensitive fragments when the colonies were derived form the end joining product of replicating NHOK, indicating that the end joining activity in these cells was remarkably accurate. However, the colonies derived from the end joining product of senescing NHOK yielded a significantly elevated number of EcoR I-resistant fragments. We obtained consistent results in the tested three strains of senescing NHOK. Thus, the frequency of abnormal end joining activity was markedly increased in NHOK during replicative senescence without donor variation. To understand the details of sequence alteration in the abnormal end joining product, sequence analysis was performed with the seven different EcoR I-resistant fragments derived from the senescing NHOK (03-1) at PD 26. We found that all tested fragments showed 5–29 bp deletion at the end joining site without apparent microhomologies, and two of the seven tested fragments showed the identical 5 bp deletion (data not shown). These results suggested that the abnormal end joining activity in senescing NHOK represented non-microhomology-mediated DNA alterations at the end joining site. 3.2. End joining of blunt DNA ends occurred with increased frequency of errors in NHOK To determine the effects of senescence on end joining of blunt DNA ends, pCR2.1 plasmid was linearized with EcoR V at the unique restriction site, and the end joining reaction was performed with exponentially replicating (PD 14) and senescing (PD 26) cultures of NHOK. PCR amplification of the expected ffi573 bp fragment was found from the end joining reaction of both NHOK cultures, and the efficiency of the end joining activity appeared to be independent of the
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Table 1 Frequency of abnormal end joining in NHOK Age/gender
NHOK strain
Population doublings (PDs)
Frequency of abnormal end joining EcoR I restriction
EcoR V restriction
53F
03-2
43M
02-1
16F
02-7
14 26 15 34 17 38
0/55 7/70 0/52 3/50 1/59 4/44
8/46 30/49 2/51 17/55 6/41 24/48
0% 10% 0% 6% 1.7% 9.1%
17.4% 61.2% 3.2% 30.3% 14.6% 50%
Fig. 2. Abnormal end joining was further increased for blunt DNA ends. To determine the efficiency and accuracy of end joining activities for blunt DSBs, pCR2.1 plasmid was linearized with EcoR V and incubated with whole cell extracts of NHOK at PD 14 and PD 16. The efficiency of end joining was similar in both cultures, as evinced in the intensity of the PCR fragment (A), but abnormal end joining of NMHEJ type were detected in the exponentially replicating culture and further increased in the senescent ones (B).
replication status (Fig. 2A). Thus, end joining of blunt DNA ends occurred without gross sequence alteration in both replicating and senescing NHOK. However, when the 573 bp PCR fragment was cloned into pcDNA3.1/V5-His TOPO plasmid, PCR-amplified and treated with the restriction enzyme, the number of EcoR V-resistant clones was markedly high, even in replicating NHOK and was further increased in senescing NHOK (Fig. 2B, Table 1). Sequence analysis of the EcoR V-resistant PCR fragments revealed the presence of variety of base pair deletions, ranging from 2 to 19 bp, at the end joining site (data not shown). These results suggest that end joining of blunt DNA DSB lesions are more error-prone than that of cohesive lesions and that the frequency of end joining errors for blunt lesions was further increased by cellular senescence in NHOK.
4. Discussion We have utilized a novel PCR-based in vitro assay to determine the efficiency and accuracy of end joining in replicating and senescing NHOK. The efficiency of end
joining was quantitated by comparing the intensities of the PCR fragments of the expected size. The accuracy of end joining was determined by noting any amplification with unexpected size, representing large deletion or insertion at the end joining site, and by testing for the restoration of the original restriction sequences. This method allowed for detection of abnormal end joining products in NHOK, which occurred primarily through two apparently independent pathways, microhomology-mediated (MHEJ) and nonmicrohomology-mediated end joining (NMHEJ). End joining of DSBs proceeded with a similar level of efficiency in replicating and senescing NHOK. End joining in senescing cells led to increased frequencies of NMHEJ errors of minor nucleotide sequence alterations at the end joining sites without apparent gross sequence deletions/ insertions. The frequency of NMHEJ errors was further elevated in end joining of blunt DSBs (EcoR V-digested) compared to those with cohesive ends (EcoR I-digested). Sequence analysis of the NMHEJ products of both EcoR Iand EcoR V-restricted DNA showed absence of microhomologies, indicating that the end joining errors stemming from MHEJ were absent in NHOK regardless of the replication status and the DSB type.
M.K. Kang et al. / Mechanisms of Ageing and Development 126 (2005) 475–479
Our results indicate that the innate DSB repair mechanism, which is generally considered to be beneficial for cell survival, may be mutagenic in senescing but not exponentially replicating NHOK, possibly contributing to genetic instability and aging. This finding is in keeping with the previous reports showing decreased fidelity of end joining in HDF during cellular senescence and accumulation of unrepairable DSBs in senescent murine cells (Sedelnikova et al., 2004; Seluanov et al., 2004). Thus, cellular senescence appears to result, in part, from accumulation of DSBs and abnormal end joining products culminating into unstable genome. This notion is supported by the findings that progerial syndromes also result from defects in Werner or Bloom DNA helicases, which are necessary for efficient DSB repair (Langland et al., 2002; Chen et al., 2003). Abnormal end joining in senescing NHOK may result from lack of one or more of the components in DNA repair machineries, including those for DSB repair. Our recent microarray-based gene expression profiling showed significantly reduced expression of genes, i.e., XRCC3, ERCC1, MLH1, MSH2, MRE11B, BRCA1 and BRCA2, involved in DNA repair mechanisms in senescing NHOK (unpublished observation). Therefore, alteration of DNA repair activities associated with cellular senescence probably involves multiple DNA repair pathways and not limited to DSB repair shown in this report. Under such circumstances, we expect to find accumulation of mutations and genetic instability during cellular senescence. Senescence may then be viewed as a protective mechanism against propagation of abnormal DNA elements. However, genetic instability may also allow senescing cells to acquire the genetic alterations necessary to escape from the senescence block and to re-enter the cell cycle, as recently demonstrated in Beausejour et al. (2003). Accordingly, accumulation of senescence cells in situ during postmaturational aging (Dimri et al., 1995) may explain the exponentially increasing risk for cancer with organismic aging.
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Acknowledgement This work was supported in part by the grants (DE14635, DE14147, DE15316) funded by the National Institute of Dental and Craniofacial Research (NIDCR). References Beausejour, C.M., Krtolica, A., Galimi, F., Narita, M., Lowe, S.W., Yaswen, P., Campisi, J., 2003. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 22, 4212–4222. Chen, L., Huang, S., Lee, L., Davalos, A., Schiestl, R.H., Campisi, J., Oshima, J., 2003. WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair. Aging Cell 2, 191– 199. Dimri, G.P., Lee, X., Basile, G., Ascosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., Peacocke, M., Campisi, J., 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363–9367. Kang, M.K., Bibb, C., Baluda, M.A., Rey, O., Park, N.-H., 2000. In vitro replication and differentiation of normal human oral keratinocytes. Exp. Cell Res. 258, 288–297. Kang, M.K., Kameta, A., Shin, K.-H., Baluda, M.A., Park, N.-H., 2004. Replicative senescence of normal human oral keratinocytes is associated with loss of telomerase activity and hTERT expression followed by limited telomere shortening. J. Cell. Physiol. 199, 264–270. Langland, G., Elliott, J., Li, Y., Creaney, J., Dixon, K., Groden, J., 2002. The BLM helicase is necessary for normal DNA double-strand break repair. Cancer Res. 62, 2766–2770. Sedelnikova, O.A., Horikawa, I., Zimonjic, D.B., Popescu, N.C., Bonner, W.M., Barrett, J.C., 2004. Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks. Nat. Cell Biol. 6, 168–170. Seluanov, A., Mittelman, D., Pereira-Smith, O.M., Wilson, J.H., Gorbunova, V., 2004. DNA end joining becomes less efficient and more errorprone during cellular senescence. Proc. Natl. Acad. Sci. USA 101, 7624–7629. Shin, K.-H., Kang, M.K., Ahn, J.-H, Lim, P.K., Yip, F.K., Yochim, J.M., Baluda, M.A., Park, N.-H. Abnormal DNA double strand break repair in human cancer cells: implication of p53 in the fidelity of DNA endjoining. Submitted for publication.
Normal human oral keratinocytes demonstrate abnormal DNA end joining activity during replicative senescence Mo K. Kang1, Ki-Hyuk Shin1, Felix K. Yip, No-Hee Park* School of Dentistry, University of California, Los Angeles, UCLA, CHS 53-038, 10833 Le Conte Avenue, Los Angeles, CA 90095-1668, USA Received 9 July 2004; received in revised form 30 September 2004; accepted 13 October 2004 Available online 9 December 2004
Abstract Repair of DNA double-strand breaks (DSBs) is critical for the maintenance of cellular genetic integrity. DSBs are repaired by cellular end joining activity, which could proceed with varying degrees of accuracy. Abnormal end joining may lead to an accumulation of mutations and contribute to genetic instability and cellular aging. In the present study, we compared the efficiency and accuracy of end joining activities in exponentially replicating and senescing normal human oral keratinocytes (NHOK). We developed an in vitro end joining assay utilizing a plasmid linearized with a unique EcoR I or EcoR V restriction site. The efficiency of end joining was determined by PCR with primers that could amplify the fragment containing the end joining site. The accuracy of end joining was assessed by determining whether the original EcoR I site was restored after end joining. Both replicating and senescing cultures of NHOK yielded a similar level of end joining efficiency, which was noted by the similar intensity of PCR amplification. However, the frequency of end joining errors was significantly elevated in NHOK during replicative senescence. Senescing NHOK could thus accumulate abnormal end joining products, which might contribute to cellular aging and cancer. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Keratinocytes; Senescence; End joining; Cancer
1. Introduction Accumulation of DSBs or abnormal DSB repair products may contribute to genetic instability and aging process. A recent study demonstrated that senescing human fibroblasts and aging mice cells accumulate unrepairable DSBs (Sedelnikova et al., 2004), suggesting that DSBs and resulting genetic instability have a causal role in cellular and organismic aging. Also, human diploid fibroblasts (HDFs) showed decreased efficiency and increased frequency of errors in end joining activity during replicative senescence further supporting the important role of DSB and abnormal DSB repair in aging (Seluanov et al., 2004). Recently, we developed a PCR-based assay for end joining activity in vitro, using whole cell lysates and pCR2.1
plasmid DNA linearized with unique EcoR I or EcoR V restriction enzyme. We employed this method to quantitatively compare the efficiency and the error frequency in end joining activity of normal human oral keratinocytes (NHOK) during serial subculture. Exponentially replicating and senescing NHOK demonstrated a similar level of end joining efficiency, while the frequency of end joining errors was found to be significantly elevated in NHOK during senescence. Thus, loss of end joining fidelity and consequent accumulation of abnormal DSB repair products may contribute to the onset of senescence in these cells.
2. Materials and methods 2.1. Cells and culture conditions
* Corresponding author. Tel.: +1 310 206 6063; fax: +1 310 794 7734. E-mail address: [email protected] (N.-H. Park). 1 Contributed equally to this work.
Primary NHOK cultures from three donors (00-4, 00-5 and 00-6) were prepared from separated epithelial tissue and
0047-6374/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mad.2004.10.002
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serially subcultured in keratinocyte growth medium (KGM, Cambrex Corp., East Rutherford, NJ, USA) containing 0.15 mM Ca++ as described previously (Kang et al., 2000). For each passage, 105 cells were plated in 100 mm culture dish and transferred when the culture reached 70% confluence. Another NHOK culture (01-7) was maintained in a feeder cell system with NIH 3T3 fibroblasts in serumcontaining medium (Kang et al., 2004). Two million mitomycin C-treated 3T3 cells and 3 105 NHOK were plated per 100 mm dish and the cells were allowed to proliferate to 70% confluence. At the time of transfer, the 3T3 cells were removed with 0.02% EDTA in 1 phosphatebuffered saline, and NHOK were trypsinized and replated on fresh feeder cells. Under these conditions, NHOK exhibited the typical first-order replication kinetics, albeit the replication potential was influenced by the culture conditions (Kang et al., 2004).
2.2. In vitro DNA end joining assay Cells were collected and washed three times in ice-cold phosphate-buffered saline (PBS). The cells were lysed by incubation for 30 min at 4 8C in lysis buffer (1% Triton X100; 150 mM NaCl; 10 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA, pH 8.0; 0.2 mM sodium ortho-vanadate). The cell lysates were cleared by centrifugation at 8000 g for 10 min at 4 8C. The pCR2.1-TOPO plasmid (Invitrogen, Carlsbad, CA, USA) was linearized by restriction with EcoR I to generate cohesiveness. The complete digestion was confirmed by agarose gel electrophoresis. The linearized DNA was subjected to phenol/chloroform extraction, ethanol-precipitated, and dissolved in sterilized water. The in vitro DNA end joining reactions (20 ml) were performed with 5 mg total cellular extract and 10 ng linearized plasmid in the presence of 4 ml of 50% polyethyleneglycol (PEG; Sigma) and 2 ml of 10 ligase buffer (300 mM Tris–HCl, pH 7.8; 100 mM KC1; 100 mM DTT and 10 mM ATP) at 37 8C for 2 h. After the end joining reaction, PCR reaction was performed with 3 ml end joining reaction using M13 reverse primer (50 CAGGAAACAGCTATGAC-30 ) and M13 forward primer (50 -GTAAAACGACGGCCAG-30 ) to amplify rejoined DNA. The PCR amplification was allowed for 30 cycles at 95 8C for 30 s, 60 8C for 30 s, and 70 8C for 30 s. PCR products were separated in 2% agarose gel and visualized by staining with ethidium bromide. Accurate end joining of the plasmid linearized with EcoR I would yield 186 bp PCR fragments. The above PCR condition allowed for exponential amplification of the DNA templates. Blunt ended DNA was prepared by complete digestion of the pCR2.1/GPR1 plasmid with EcoR V (New England Biolabs). This plasmid contained an additional 371 bp of partial GPR1 fragment cloned into a cloning site (overhanging 30 deoxythymidine residues) outside the unique EcoR V restriction site within the pCR2.1 backbone.
Accurate end joining of the plasmid linearized with EcoR V was amplified as a band of 573 bp. The efficiency of end joining was determined by noting the intensity of the 186 or 573 bp PCR fragment and was quantitated using Scion image analysis software (Scion Corp., Frederek, MD, USA). The accuracy of end joining was assessed by cloning the PCR fragment into pcDNA3.1/ V5-His TOPO plasmid (Invitrogen). The resulting ligation products were introduced into Eschericia coli strain TOP10 (Invitrogen). Subsequently, single colony PCR was performed using the M13 primer set. The PCR product was digested with EcoR I or EcoR V and electrophoresed in 2% agarose gel to compare the restriction profiles. The PCR products, which were resistant to the EcoR I or EcoR V digestion, were considered as abnormal end joining with sequence alteration. The PCR products sensitive to the enzyme digestion represented the precise end joining activity. The frequency of abnormal end joining was determined by noting the number of E. coli colonies, which gave rise to the PCR product resistant to the enzyme digestion. 2.3. DNA sequencing analysis To determine the exact sequence alterations in abnormal end joining, the PCR fragments resistant to either EcoR I or EcoR V digestion were sequenced directly with a Taq dideoxy terminator cycle sequencing kit on an ABI 377 automatic DNA sequencer (Perkin–Elmer; UCLA Sequencing Core Facility).
3. Results 3.1. Senescing NHOK demonstrated increased frequency of abnormal end joining activity We developed a PCR-based in vitro end joining assay for quantitative determination of both precise and abnormal end joining activities (Shin et al., manuscript submitted). pCR2.1 plasmid was linearized at the unique EcoR I restriction site to create cohesive DNA ends and incubated with whole cell lysates. Plasmid end joining was detected by PCR amplification of the plasmid sequences containing the EcoR I site, yielding 186 bp fragments. To demonstrate the efficiency of (or lack thereof) end joining simply by DNA end annealing event, we also performed the in vitro end joining assay in the absence of cell lysate. We found that there was no amplification of the substrate DNA without exposure to the cell lysate (Shin et al., manuscript submitted). This finding indicates complete digestion of the plasmid DNA and lack of false amplification resulting from sticky end annealing. We also performed the in vitro DNA end joining assay and varied (1) the amount of cell lysate, (2) number of PCR amplification cycles, or (3) amount of linearized DNA substrate. We found that the level
M.K. Kang et al. / Mechanisms of Ageing and Development 126 (2005) 475–479
477
Fig. 1. Senescent NHOK demonstrate abnormal end joining activity in vitro. Whole cell extracts of NHOK at PD 14 and PD 16 were incubated with pCR2.1 plasmid DNA linearized with EcoR I. The level of end joining was determined by PCR amplification of regions containing the restriction site (A). Restoration of the original EcoR I restriction sequence was determined by isolating the PCR fragments from (A) and performing single-colony PCR followed by EcoR I digestion (B). The doublets indicate EcoR I-sensitive fragment, while the high molecular weight single band was resistant to the enzyme digestion.
of DNA substrate amplification using the PCR conditions described in this study is quantitatively correlated to the above variables (Shin et al., manuscript submitted). Therefore, efficiency of cellular end joining activity would be correlated to the intensity of PCR amplification, while the accuracy of end joining could be assessed by the molecular size of the PCR products and restoration of the EcoR I restriction sequence. To determine the effects of replicative senescence on the efficiency and accuracy of end joining activity in NHOK, the in vitro end joining assay was performed with exponentially replicating and senescing culture of NHOK (Fig. 1A). Both NHOK cultures at PD 14 and PD 26 demonstrated a similar level of end joining efficiency and the reaction yielded the expected 186 bp fragment. Also, end joining in NHOK proceeded without gross sequence alteration of the end joining site regardless of the cellular replication status. The 186 bp PCR fragment presumably contained a variety of end joining reaction products, which may possess errors not detectable by agarose gel electrophoresis. Therefore, the accuracy of end joining was also determined by restoration of the EcoR I site in the individual PCR fragments. The 186 bp fragments obtained from end joining activity of exponentially replicating and senescing NHOK were excised from the agarose gel and cloned into pcDNA3.1/V5-His TOPO plasmid. Competent E. coli was transformed with the resulting mixture of constructs and single colony PCR amplification was performed using the M13 primers. The PCR products were digested with EcoR I and electrophoresed in 2% agarose (Fig. 1B). The colonies, which gave rise to EcoR I-resistant PCR fragments were scored as representing abnormal end joining activity. Using this method, we have tested the accuracy of end joining activity in three different strains of NHOK (03-1, 02-1, and 02-7) at the exponentially replicating and senescing phases
of replication (Table 1). We found that the vast majority of the colonies yielded EcoR I-sensitive fragments when the colonies were derived form the end joining product of replicating NHOK, indicating that the end joining activity in these cells was remarkably accurate. However, the colonies derived from the end joining product of senescing NHOK yielded a significantly elevated number of EcoR I-resistant fragments. We obtained consistent results in the tested three strains of senescing NHOK. Thus, the frequency of abnormal end joining activity was markedly increased in NHOK during replicative senescence without donor variation. To understand the details of sequence alteration in the abnormal end joining product, sequence analysis was performed with the seven different EcoR I-resistant fragments derived from the senescing NHOK (03-1) at PD 26. We found that all tested fragments showed 5–29 bp deletion at the end joining site without apparent microhomologies, and two of the seven tested fragments showed the identical 5 bp deletion (data not shown). These results suggested that the abnormal end joining activity in senescing NHOK represented non-microhomology-mediated DNA alterations at the end joining site. 3.2. End joining of blunt DNA ends occurred with increased frequency of errors in NHOK To determine the effects of senescence on end joining of blunt DNA ends, pCR2.1 plasmid was linearized with EcoR V at the unique restriction site, and the end joining reaction was performed with exponentially replicating (PD 14) and senescing (PD 26) cultures of NHOK. PCR amplification of the expected ffi573 bp fragment was found from the end joining reaction of both NHOK cultures, and the efficiency of the end joining activity appeared to be independent of the
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Table 1 Frequency of abnormal end joining in NHOK Age/gender
NHOK strain
Population doublings (PDs)
Frequency of abnormal end joining EcoR I restriction
EcoR V restriction
53F
03-2
43M
02-1
16F
02-7
14 26 15 34 17 38
0/55 7/70 0/52 3/50 1/59 4/44
8/46 30/49 2/51 17/55 6/41 24/48
0% 10% 0% 6% 1.7% 9.1%
17.4% 61.2% 3.2% 30.3% 14.6% 50%
Fig. 2. Abnormal end joining was further increased for blunt DNA ends. To determine the efficiency and accuracy of end joining activities for blunt DSBs, pCR2.1 plasmid was linearized with EcoR V and incubated with whole cell extracts of NHOK at PD 14 and PD 16. The efficiency of end joining was similar in both cultures, as evinced in the intensity of the PCR fragment (A), but abnormal end joining of NMHEJ type were detected in the exponentially replicating culture and further increased in the senescent ones (B).
replication status (Fig. 2A). Thus, end joining of blunt DNA ends occurred without gross sequence alteration in both replicating and senescing NHOK. However, when the 573 bp PCR fragment was cloned into pcDNA3.1/V5-His TOPO plasmid, PCR-amplified and treated with the restriction enzyme, the number of EcoR V-resistant clones was markedly high, even in replicating NHOK and was further increased in senescing NHOK (Fig. 2B, Table 1). Sequence analysis of the EcoR V-resistant PCR fragments revealed the presence of variety of base pair deletions, ranging from 2 to 19 bp, at the end joining site (data not shown). These results suggest that end joining of blunt DNA DSB lesions are more error-prone than that of cohesive lesions and that the frequency of end joining errors for blunt lesions was further increased by cellular senescence in NHOK.
4. Discussion We have utilized a novel PCR-based in vitro assay to determine the efficiency and accuracy of end joining in replicating and senescing NHOK. The efficiency of end
joining was quantitated by comparing the intensities of the PCR fragments of the expected size. The accuracy of end joining was determined by noting any amplification with unexpected size, representing large deletion or insertion at the end joining site, and by testing for the restoration of the original restriction sequences. This method allowed for detection of abnormal end joining products in NHOK, which occurred primarily through two apparently independent pathways, microhomology-mediated (MHEJ) and nonmicrohomology-mediated end joining (NMHEJ). End joining of DSBs proceeded with a similar level of efficiency in replicating and senescing NHOK. End joining in senescing cells led to increased frequencies of NMHEJ errors of minor nucleotide sequence alterations at the end joining sites without apparent gross sequence deletions/ insertions. The frequency of NMHEJ errors was further elevated in end joining of blunt DSBs (EcoR V-digested) compared to those with cohesive ends (EcoR I-digested). Sequence analysis of the NMHEJ products of both EcoR Iand EcoR V-restricted DNA showed absence of microhomologies, indicating that the end joining errors stemming from MHEJ were absent in NHOK regardless of the replication status and the DSB type.
M.K. Kang et al. / Mechanisms of Ageing and Development 126 (2005) 475–479
Our results indicate that the innate DSB repair mechanism, which is generally considered to be beneficial for cell survival, may be mutagenic in senescing but not exponentially replicating NHOK, possibly contributing to genetic instability and aging. This finding is in keeping with the previous reports showing decreased fidelity of end joining in HDF during cellular senescence and accumulation of unrepairable DSBs in senescent murine cells (Sedelnikova et al., 2004; Seluanov et al., 2004). Thus, cellular senescence appears to result, in part, from accumulation of DSBs and abnormal end joining products culminating into unstable genome. This notion is supported by the findings that progerial syndromes also result from defects in Werner or Bloom DNA helicases, which are necessary for efficient DSB repair (Langland et al., 2002; Chen et al., 2003). Abnormal end joining in senescing NHOK may result from lack of one or more of the components in DNA repair machineries, including those for DSB repair. Our recent microarray-based gene expression profiling showed significantly reduced expression of genes, i.e., XRCC3, ERCC1, MLH1, MSH2, MRE11B, BRCA1 and BRCA2, involved in DNA repair mechanisms in senescing NHOK (unpublished observation). Therefore, alteration of DNA repair activities associated with cellular senescence probably involves multiple DNA repair pathways and not limited to DSB repair shown in this report. Under such circumstances, we expect to find accumulation of mutations and genetic instability during cellular senescence. Senescence may then be viewed as a protective mechanism against propagation of abnormal DNA elements. However, genetic instability may also allow senescing cells to acquire the genetic alterations necessary to escape from the senescence block and to re-enter the cell cycle, as recently demonstrated in Beausejour et al. (2003). Accordingly, accumulation of senescence cells in situ during postmaturational aging (Dimri et al., 1995) may explain the exponentially increasing risk for cancer with organismic aging.
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Acknowledgement This work was supported in part by the grants (DE14635, DE14147, DE15316) funded by the National Institute of Dental and Craniofacial Research (NIDCR). References Beausejour, C.M., Krtolica, A., Galimi, F., Narita, M., Lowe, S.W., Yaswen, P., Campisi, J., 2003. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J. 22, 4212–4222. Chen, L., Huang, S., Lee, L., Davalos, A., Schiestl, R.H., Campisi, J., Oshima, J., 2003. WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repair. Aging Cell 2, 191– 199. Dimri, G.P., Lee, X., Basile, G., Ascosta, M., Scott, G., Roskelley, C., Medrano, E.E., Linskens, M., Rubelj, I., Pereira-Smith, O., Peacocke, M., Campisi, J., 1995. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363–9367. Kang, M.K., Bibb, C., Baluda, M.A., Rey, O., Park, N.-H., 2000. In vitro replication and differentiation of normal human oral keratinocytes. Exp. Cell Res. 258, 288–297. Kang, M.K., Kameta, A., Shin, K.-H., Baluda, M.A., Park, N.-H., 2004. Replicative senescence of normal human oral keratinocytes is associated with loss of telomerase activity and hTERT expression followed by limited telomere shortening. J. Cell. Physiol. 199, 264–270. Langland, G., Elliott, J., Li, Y., Creaney, J., Dixon, K., Groden, J., 2002. The BLM helicase is necessary for normal DNA double-strand break repair. Cancer Res. 62, 2766–2770. Sedelnikova, O.A., Horikawa, I., Zimonjic, D.B., Popescu, N.C., Bonner, W.M., Barrett, J.C., 2004. Senescing human cells and ageing mice accumulate DNA lesions with unrepairable double-strand breaks. Nat. Cell Biol. 6, 168–170. Seluanov, A., Mittelman, D., Pereira-Smith, O.M., Wilson, J.H., Gorbunova, V., 2004. DNA end joining becomes less efficient and more errorprone during cellular senescence. Proc. Natl. Acad. Sci. USA 101, 7624–7629. Shin, K.-H., Kang, M.K., Ahn, J.-H, Lim, P.K., Yip, F.K., Yochim, J.M., Baluda, M.A., Park, N.-H. Abnormal DNA double strand break repair in human cancer cells: implication of p53 in the fidelity of DNA endjoining. Submitted for publication.