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Telomere shortening does not occur during postmaturational aging in situ in normal human oral fibroblasts
Mechanisms of Ageing and Development 124 (2003) 873 /876 www.elsevier.com/locate/mechagedev
Telomere shortening does not occur during postmaturational aging in situ in normal human oral fibroblasts Mo K. Kang a,b, Ayako Kameta b, Marcel A. Baluda b,c, No-Hee Park a,b,* a
UCLA School of Dentistry, University of California, CHS 53-038, 10833 Le Conte Ave., Los Angeles, CA 90095-1668, USA b Dental Research Institute, University of California, Los Angeles, CA, USA c School of Medicine, University of California, Los Angeles, CA, USA Received 16 December 2002; received in revised form 14 May 2003; accepted 14 May 2003
Abstract We investigated whether the telomere length, i.e. mean terminal restriction fragment (TRF) length, decreases during in situ aging in normal human oral fibroblasts (NHOF). For this purpose, NHOF cultures were established from 50 different donors and tested after 14 population doublings (PD) when the cells were replicating exponentially. Telomere-specific Southern blotting and digital quantitation showed that the mean (9/standard error (S.E.)) TRF length of all tested cultures was 7.729/0.17 kbps. The plot of TRF mean length versus donor age showed high variability in individual length with an apparent average decline of /7.8 bp per year of age, which was not statistically significant (r /0.11; P /0.1). These data indicate that telomere shortening does not occur during donor aging in situ, and therefore, is not physiologically relevant for NHOF. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Telomere; Telomerase; Fibroblasts; Senescence; Aging
The telomere shortening hypothesis of replicative senescence was originally proposed based on the finding that telomeric DNA shortens during aging of human diploid fibroblasts (HDF) in vitro and in vivo (Harley et al., 1990). While telomere shortening has been well documented during cellular replication and aging in a wide variety of cells and tissues (Hastie et al., 1990; Vaziri et al., 1993; Allsopp et al., 1995; Butler et al., 1998; Kang et al., 2002), there is controversy about in vivo telomere shortening and senescence of HDF (Cristofalo et al., 1998; Tresini et al., 1999; Mondello et al., 1999). We have previously presented some evidence suggesting the absence of telomere shortening in normal human oral fibroblasts (NHOF) during in situ aging (Kang et al., 2002). The current study was undertaken as an extension of our previous finding using NHOF cultures established from 50 healthy donors ranging in age from 11 to 75-years-old. This large sample number was utilized to settle statistically
* Corresponding author. Tel.: /1-310-206-6063; fax: /1-310-7947734. E-mail address: [email protected] (N.-H. Park).
the effect of postmaturational aging in situ on telomere length in HDF. Primary NHOF cultures were established from explants of oral mucosal tissues of approximately 5 /5 mm2 size, according to the method described elsewhere (Kang et al., 1998). The cultures were maintained in 4:1 mixture of Dulbecco’s modified minimum essential medium (DMEM) and Medium 199 (Invitrogen, Carlesbad, CA) containing 10% fetal bovine serum in 5% CO2 at 37 8C. The cells were allowed to replicate until the 100-mm dishes became 70 /80% confluent. The mean (9/standard error (S.E.)) population doublings (PD) at the time of harvest was 13.629/0.17, at which time the cultures consisted entirely of exponentially replicating NHOF. Genomic DNA was isolated from these cells and digested to completion with HinF I and Rsa I. 10 mg of the restricted DNA fragments were separated by pulse-field gel electrophoresis using the FIGE Mapper Field Inversion System (Bio-Rad, Hercules, CA). We performed telomere-specific Southern hybridization using a (TTAGGG)16 probe as described previously (Kang et al., 1998) (Fig. 1). The terminal restriction fragment (TRF) length of individual NHOF cultures
0047-6374/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0047-6374(03)00145-3
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M.K. Kang et al. / Mechanisms of Ageing and Development 124 (2003) 873 /876
Fig. 1. Telomere-specific Southern blots of 50 different NHOF strains. Exponentially replicating NHOF cultures were harvested and genomic DNA was isolated. 10 mg of DNA was completely restricted with HinF I and Rsa I and separated by pulse-field gel electrophoresis. Telomeric sequences were detected using 32P-[dCTP]-labeled (TTAGGG)16 probe. The mean TRF length of individual NHOF was quantitated using IMAGEQUANT software (Molecular Dynamix, Sunnyvale, CA), according to the method described elsewhere (Kang et al., 2002). The panels A /E show five different blots, and the panel F shows a direct comparison between five young ( B/20-years-old) and five old ( /60-years-old) donors.
ranged from 5.83 to 12.15 kbps, and the mean (9/S.E.) of all cultures was 7.729/0.17 kbps. When the individual TRF length was plotted against the donor age, the trend line representing the least-squares fit of all data appeared to show a decrease of TRF length by 7.8 bp per year (Fig. 2). However, the negative correlation between the TRF length and the donor age (correlation coefficient, r /0.11; P /0.1) was not statistically significant. The culture cells were harvested within a narrow range of PDs, i.e. 13.629/0.17, and there was not a statistically significant correlation (r /0.06; P / 0.1) between the PD and TRF length. These results demonstrated that telomere length in NHOF was not altered in situ by donor aging. Even if we accepted a TRF length decrease of 7.8 bp per year, it would take 422 years to reach 4.8 kbps, at which serially subcultured NHOF entered senescence in vitro (Fig. 3). Although the telomeric length of senescent NHOF in situ is not known, our results showed that a TRF length above 4.8 kbps was compatible with cellular replication. Therefore, the critical telomere length does not appear to limit the replication of NHOF in situ during the physiological life span of an individual. A recent study showed that senescence is caused by altered telomere function and not by complete loss of telomeres per se (Karlseder et al., 2002). The same report also
Fig. 2. Telomere length was maintained in NHOF during postmaturational aging in situ. The mean TRF length of all NHOF strains was plotted against the donor age. The trend line was calculated by the least-squares fit of all data and was represented by the equation, y / / 0.0078m/8.0878, where y denotes the TRF length in kbps and m the mean TRF length. Extrapolation of the line indicated a TRF length of 8.09 kbps at birth. However, the negative correlation between the TRF length and the donor age was not statistically significant (correlation coefficient, r / /0.11; P /0.1). Also, there was no significant difference in the TRF length between the male (') and female (D) donors.
M.K. Kang et al. / Mechanisms of Ageing and Development 124 (2003) 873 /876
875
Fig. 4. Comparison of TRF length between NHOF and NHOK. The TRF length of all NHOF (j) samples was separated into seven 10 year-age groups and compared with that of NHOK (I) in the same age groups. The data for NHOK were derived from our previous publication (Kang et al., 2002). The bar represents S.E. of the mean.
Fig. 3. The TRF length shortens progressively in serially subcultured NHOF in vitro. One NHOF strain was serially subcultured until the cells spontaneously arrested replication during the senescence phase. (A) Telomere-specific Southern blotting was performed with 10 mg genomic DNA as described in Fig. 2 to compare the TRF length at increasing PD levels. (B) The mean TRF length at each PD level was quantitated and plotted against the PD number demonstrating a linear negative correlation (r /0.98; P B/0.001). The rate of shortening calculated from the least-squares fit was /53 bp per PD.
suggested that altered telomere function, i.e. loss of telomere protection, also required shortening of telomeric DNA. Thus, there is a remote possibility of agedependent alteration of telomere function in NHOF, which exhibited no telomere shortening during donor aging, although such effect deserves further investigation. The difference between the telomere length dynamics of NHOF in vitro and in situ can best be explained if replication of NHOF is normally repressed in situ. This explanation is supported by our previous report showing that the TRF length of NHOF was significantly higher than that of normal human oral keratinocytes (NHOK), which undergo constant cycles of replication, differentiation, and regeneration in situ (Fig. 4) (Kang et
al., 2002). The above notion is also in keeping with the finding that the proliferative capacity of HDF is not altered by donor age (Cristofalo et al., 1998). One may argue that mesenchymal stem cells may be present in the pool of HDF explanted from aged donors, although no such data have been reported. Repression of NHOF replication in situ appears to be true only during postmaturational aging. Extrapolation of the data presented in Fig. 2 to year zero yielded a TRF length of 8.09 kbps at birth. This value is markedly smaller than the reported telomeric length of 10 /12 kbps in normal human somatic cells at birth (de Lange et al., 1990; Vaziri et al., 1993), indicating a period of rapid telomere shortening in NHOF during the early part of an individual’s life span. Based on these findings, we conclude that NHOF undergo massive cell proliferation during organismic development, resulting in shortening of telomeric DNA, which is subsequently maintained at constant length during postmaturational aging in situ. A recent report suggested that the standard method of preparing skin fibroblast cultures from tissue explants selected for the fastest growing cell population and was not fully representative of the cells in the tissues (Balin et al., 2002). It is also possible that selection for rapidly dividing NHOF have occurred during the initial period of culture in our study. However, we did not find statistically significant difference in time required to establish the initial culture between the tissues obtained from the younger donors (249/6 days for donors less than 50 years old) and those from the older ones (269/8 days for donors older than 50 years old; P /0.1). Also, there was no significant correlation between time required to establish the initial culture and TRF length of the corresponding NHOF strain (r /0.17; P /0.1). Thus, our data argue against the possibilities that
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rapidly dividing cells were more abundant in the tissues obtained from the younger donors compared with those from the older ones and that relatively long TRFs found in older donors reflected selection of rapidly dividing subpopulation of cells in the tissues. It is important to note the difference between NHOF and NHOK in their telomere dynamics and replicative capacities in situ and in vitro. As illustrated in Fig. 4, the difference in the TRF length between the two cell types increased with donor aging because telomere length in NHOK progressively shortened. Interestingly, serial cultivation of these cells showed opposite effects; telomere DNA rapidly shortened in NHOF but was maintained at constant length in replicating NHOK during in vitro passage (Kang et al., 1998). This difference in vitro was attributed to the finding that telomerase activity was present in replicating NHOK and not in NHOF (Kang et al., 1998). From an evolutionary standpoint, it is also conceivable that telomerase activity is necessary for NHOK, which undergo continuous cycles of replication in situ, while the enzyme activity is not needed for NHOF probably because these cells do not replicate and shorten their telomeres in situ. Our previous data also suggested that telomere shortening in NHOK in situ resulted from an accumulation of short telomeres in aged donors (Kang et al., 2002). During cellular immortalization and carcinogenesis, cells with short telomeres may have higher probability of transformation through chromosomal instability and resulting genetic alterations. The absence of cellular replication and telomere shortening in NHOF and their occurrence in NHOK during postmaturational aging may be critically linked to the well-known epidemiological fact that the incidence of squamous cell carcinoma derived from the oral epithelium accounts for more than 90% of all malignant cancer in the human oral cavity (Lucas, 1977).
Acknowledgements This work was supported in part by the grants (DE14635-01 and DE14147) funded by the National Institute of Dental and Craniofacial Research (NIDCR). MKK is a recipient of the Educator Fellow-
ship Award from the American Association of Endodontists.
References Allsopp, R.C., Chang, E., Kashefi-Aazam, M., Rogaev, E.I., Piatyszek, M.A., Shay, J.W., Harley, C.B., 1995. Telomere shortening is associated with cell division in vitro and in vivo. Exp. Cell Res. 220, 194 /200. Balin, A.K., Fisher, A.J., Anzelone, M., Leong, I., Allen, R.G., 2002. Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages. Exp. Cell Res. 274, 275 /287. Butler, M.G., Tilburt, J., DeVries, A., Muralidhar, B., Aue, G., Hedges, L., Atkinson, J., Schwartz, H, 1998. Comparison of chromosome telomere integrity in multiple tissues from subjects at different ages. Cancer Genet. Cytogenet. 105, 138 /144. Cristofalo, V.J., Allen, R.G., Pgnolo, R.J., Martin, B.G., Beck, J.C., 1998. Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation. Proc. Natl. Acad. Sci. USA 95, 10614 /10619. de Lange, T., Shiue, L., Myers, R.M., Cox, D.R., Naylor, S.L., Killery, A.M., Varmus, H.E., 1990. Structure and variability of human chromosome ends. Mol. Cell. Biol. 10, 518 /527. Harley, C.B., Futcher, B., Greider, C.W., 1990. Telomeres shorten during aging of human fibroblasts. Nature 345, 458 /460. Hastie, N.D., Dempster, M., Dunlop, M.G., Thompson, A.M., Green, D.K., Allshire, R.C., 1990. Telomere reduction in human colorectal carcinoma and with ageing. Nature 346, 866 /868. Kang, M.K., Guo, W., Park, N.-H., 1998. Replicative senescence of normal human oral keratinocytes is associated with the loss of telomerase activity without shortening of telomeres. Cell Growth Differ. 9, 85 /95. Kang, M.K., Swee, J., Kim, R.H., Baluda, M.A., Park, N.-H., 2002. The telomeric length and heterogeneity decrease by age in normal human oral keratinocytes. Mech. Ageing Dev. 123, 585 /592. Karlseder, J., Smogorzewska, A., de Lange, T., 2002. Senescence induced by altered telomere state, not telomere loss. Science 295, 2446 /2449. Lucas, R.B., 1977. The epidemiology of oral tumors. Int. Dent. J. 27, 294 /298. Mondello, C., Petropoulou, C., Monti, D., Gonos, E.S., Franceschi, C., Nuzzo, F., 1999. Telomere length in fibroblasts and blood cells from healthy centenarians. Exp. Cell Res. 248, 234 /242. Tresini, M., Pignolo, R.J., Allen, R.G., Cristofalo, V.J., 1999. Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts. Cell Physiol. 197, 11 /17. Vaziri, H., Scha¨chter, F., Uchida, I., Wei, L., Zhu, X., Effros, R., Cohen, D., Harley, C.B., 1993. Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am. J. Hum. Genet. 52, 661 /667.
Telomere shortening does not occur during postmaturational aging in situ in normal human oral fibroblasts Mo K. Kang a,b, Ayako Kameta b, Marcel A. Baluda b,c, No-Hee Park a,b,* a
UCLA School of Dentistry, University of California, CHS 53-038, 10833 Le Conte Ave., Los Angeles, CA 90095-1668, USA b Dental Research Institute, University of California, Los Angeles, CA, USA c School of Medicine, University of California, Los Angeles, CA, USA Received 16 December 2002; received in revised form 14 May 2003; accepted 14 May 2003
Abstract We investigated whether the telomere length, i.e. mean terminal restriction fragment (TRF) length, decreases during in situ aging in normal human oral fibroblasts (NHOF). For this purpose, NHOF cultures were established from 50 different donors and tested after 14 population doublings (PD) when the cells were replicating exponentially. Telomere-specific Southern blotting and digital quantitation showed that the mean (9/standard error (S.E.)) TRF length of all tested cultures was 7.729/0.17 kbps. The plot of TRF mean length versus donor age showed high variability in individual length with an apparent average decline of /7.8 bp per year of age, which was not statistically significant (r /0.11; P /0.1). These data indicate that telomere shortening does not occur during donor aging in situ, and therefore, is not physiologically relevant for NHOF. # 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Telomere; Telomerase; Fibroblasts; Senescence; Aging
The telomere shortening hypothesis of replicative senescence was originally proposed based on the finding that telomeric DNA shortens during aging of human diploid fibroblasts (HDF) in vitro and in vivo (Harley et al., 1990). While telomere shortening has been well documented during cellular replication and aging in a wide variety of cells and tissues (Hastie et al., 1990; Vaziri et al., 1993; Allsopp et al., 1995; Butler et al., 1998; Kang et al., 2002), there is controversy about in vivo telomere shortening and senescence of HDF (Cristofalo et al., 1998; Tresini et al., 1999; Mondello et al., 1999). We have previously presented some evidence suggesting the absence of telomere shortening in normal human oral fibroblasts (NHOF) during in situ aging (Kang et al., 2002). The current study was undertaken as an extension of our previous finding using NHOF cultures established from 50 healthy donors ranging in age from 11 to 75-years-old. This large sample number was utilized to settle statistically
* Corresponding author. Tel.: /1-310-206-6063; fax: /1-310-7947734. E-mail address: [email protected] (N.-H. Park).
the effect of postmaturational aging in situ on telomere length in HDF. Primary NHOF cultures were established from explants of oral mucosal tissues of approximately 5 /5 mm2 size, according to the method described elsewhere (Kang et al., 1998). The cultures were maintained in 4:1 mixture of Dulbecco’s modified minimum essential medium (DMEM) and Medium 199 (Invitrogen, Carlesbad, CA) containing 10% fetal bovine serum in 5% CO2 at 37 8C. The cells were allowed to replicate until the 100-mm dishes became 70 /80% confluent. The mean (9/standard error (S.E.)) population doublings (PD) at the time of harvest was 13.629/0.17, at which time the cultures consisted entirely of exponentially replicating NHOF. Genomic DNA was isolated from these cells and digested to completion with HinF I and Rsa I. 10 mg of the restricted DNA fragments were separated by pulse-field gel electrophoresis using the FIGE Mapper Field Inversion System (Bio-Rad, Hercules, CA). We performed telomere-specific Southern hybridization using a (TTAGGG)16 probe as described previously (Kang et al., 1998) (Fig. 1). The terminal restriction fragment (TRF) length of individual NHOF cultures
0047-6374/03/$ - see front matter # 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/S0047-6374(03)00145-3
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M.K. Kang et al. / Mechanisms of Ageing and Development 124 (2003) 873 /876
Fig. 1. Telomere-specific Southern blots of 50 different NHOF strains. Exponentially replicating NHOF cultures were harvested and genomic DNA was isolated. 10 mg of DNA was completely restricted with HinF I and Rsa I and separated by pulse-field gel electrophoresis. Telomeric sequences were detected using 32P-[dCTP]-labeled (TTAGGG)16 probe. The mean TRF length of individual NHOF was quantitated using IMAGEQUANT software (Molecular Dynamix, Sunnyvale, CA), according to the method described elsewhere (Kang et al., 2002). The panels A /E show five different blots, and the panel F shows a direct comparison between five young ( B/20-years-old) and five old ( /60-years-old) donors.
ranged from 5.83 to 12.15 kbps, and the mean (9/S.E.) of all cultures was 7.729/0.17 kbps. When the individual TRF length was plotted against the donor age, the trend line representing the least-squares fit of all data appeared to show a decrease of TRF length by 7.8 bp per year (Fig. 2). However, the negative correlation between the TRF length and the donor age (correlation coefficient, r /0.11; P /0.1) was not statistically significant. The culture cells were harvested within a narrow range of PDs, i.e. 13.629/0.17, and there was not a statistically significant correlation (r /0.06; P / 0.1) between the PD and TRF length. These results demonstrated that telomere length in NHOF was not altered in situ by donor aging. Even if we accepted a TRF length decrease of 7.8 bp per year, it would take 422 years to reach 4.8 kbps, at which serially subcultured NHOF entered senescence in vitro (Fig. 3). Although the telomeric length of senescent NHOF in situ is not known, our results showed that a TRF length above 4.8 kbps was compatible with cellular replication. Therefore, the critical telomere length does not appear to limit the replication of NHOF in situ during the physiological life span of an individual. A recent study showed that senescence is caused by altered telomere function and not by complete loss of telomeres per se (Karlseder et al., 2002). The same report also
Fig. 2. Telomere length was maintained in NHOF during postmaturational aging in situ. The mean TRF length of all NHOF strains was plotted against the donor age. The trend line was calculated by the least-squares fit of all data and was represented by the equation, y / / 0.0078m/8.0878, where y denotes the TRF length in kbps and m the mean TRF length. Extrapolation of the line indicated a TRF length of 8.09 kbps at birth. However, the negative correlation between the TRF length and the donor age was not statistically significant (correlation coefficient, r / /0.11; P /0.1). Also, there was no significant difference in the TRF length between the male (') and female (D) donors.
M.K. Kang et al. / Mechanisms of Ageing and Development 124 (2003) 873 /876
875
Fig. 4. Comparison of TRF length between NHOF and NHOK. The TRF length of all NHOF (j) samples was separated into seven 10 year-age groups and compared with that of NHOK (I) in the same age groups. The data for NHOK were derived from our previous publication (Kang et al., 2002). The bar represents S.E. of the mean.
Fig. 3. The TRF length shortens progressively in serially subcultured NHOF in vitro. One NHOF strain was serially subcultured until the cells spontaneously arrested replication during the senescence phase. (A) Telomere-specific Southern blotting was performed with 10 mg genomic DNA as described in Fig. 2 to compare the TRF length at increasing PD levels. (B) The mean TRF length at each PD level was quantitated and plotted against the PD number demonstrating a linear negative correlation (r /0.98; P B/0.001). The rate of shortening calculated from the least-squares fit was /53 bp per PD.
suggested that altered telomere function, i.e. loss of telomere protection, also required shortening of telomeric DNA. Thus, there is a remote possibility of agedependent alteration of telomere function in NHOF, which exhibited no telomere shortening during donor aging, although such effect deserves further investigation. The difference between the telomere length dynamics of NHOF in vitro and in situ can best be explained if replication of NHOF is normally repressed in situ. This explanation is supported by our previous report showing that the TRF length of NHOF was significantly higher than that of normal human oral keratinocytes (NHOK), which undergo constant cycles of replication, differentiation, and regeneration in situ (Fig. 4) (Kang et
al., 2002). The above notion is also in keeping with the finding that the proliferative capacity of HDF is not altered by donor age (Cristofalo et al., 1998). One may argue that mesenchymal stem cells may be present in the pool of HDF explanted from aged donors, although no such data have been reported. Repression of NHOF replication in situ appears to be true only during postmaturational aging. Extrapolation of the data presented in Fig. 2 to year zero yielded a TRF length of 8.09 kbps at birth. This value is markedly smaller than the reported telomeric length of 10 /12 kbps in normal human somatic cells at birth (de Lange et al., 1990; Vaziri et al., 1993), indicating a period of rapid telomere shortening in NHOF during the early part of an individual’s life span. Based on these findings, we conclude that NHOF undergo massive cell proliferation during organismic development, resulting in shortening of telomeric DNA, which is subsequently maintained at constant length during postmaturational aging in situ. A recent report suggested that the standard method of preparing skin fibroblast cultures from tissue explants selected for the fastest growing cell population and was not fully representative of the cells in the tissues (Balin et al., 2002). It is also possible that selection for rapidly dividing NHOF have occurred during the initial period of culture in our study. However, we did not find statistically significant difference in time required to establish the initial culture between the tissues obtained from the younger donors (249/6 days for donors less than 50 years old) and those from the older ones (269/8 days for donors older than 50 years old; P /0.1). Also, there was no significant correlation between time required to establish the initial culture and TRF length of the corresponding NHOF strain (r /0.17; P /0.1). Thus, our data argue against the possibilities that
876
M.K. Kang et al. / Mechanisms of Ageing and Development 124 (2003) 873 /876
rapidly dividing cells were more abundant in the tissues obtained from the younger donors compared with those from the older ones and that relatively long TRFs found in older donors reflected selection of rapidly dividing subpopulation of cells in the tissues. It is important to note the difference between NHOF and NHOK in their telomere dynamics and replicative capacities in situ and in vitro. As illustrated in Fig. 4, the difference in the TRF length between the two cell types increased with donor aging because telomere length in NHOK progressively shortened. Interestingly, serial cultivation of these cells showed opposite effects; telomere DNA rapidly shortened in NHOF but was maintained at constant length in replicating NHOK during in vitro passage (Kang et al., 1998). This difference in vitro was attributed to the finding that telomerase activity was present in replicating NHOK and not in NHOF (Kang et al., 1998). From an evolutionary standpoint, it is also conceivable that telomerase activity is necessary for NHOK, which undergo continuous cycles of replication in situ, while the enzyme activity is not needed for NHOF probably because these cells do not replicate and shorten their telomeres in situ. Our previous data also suggested that telomere shortening in NHOK in situ resulted from an accumulation of short telomeres in aged donors (Kang et al., 2002). During cellular immortalization and carcinogenesis, cells with short telomeres may have higher probability of transformation through chromosomal instability and resulting genetic alterations. The absence of cellular replication and telomere shortening in NHOF and their occurrence in NHOK during postmaturational aging may be critically linked to the well-known epidemiological fact that the incidence of squamous cell carcinoma derived from the oral epithelium accounts for more than 90% of all malignant cancer in the human oral cavity (Lucas, 1977).
Acknowledgements This work was supported in part by the grants (DE14635-01 and DE14147) funded by the National Institute of Dental and Craniofacial Research (NIDCR). MKK is a recipient of the Educator Fellow-
ship Award from the American Association of Endodontists.
References Allsopp, R.C., Chang, E., Kashefi-Aazam, M., Rogaev, E.I., Piatyszek, M.A., Shay, J.W., Harley, C.B., 1995. Telomere shortening is associated with cell division in vitro and in vivo. Exp. Cell Res. 220, 194 /200. Balin, A.K., Fisher, A.J., Anzelone, M., Leong, I., Allen, R.G., 2002. Effects of establishing cell cultures and cell culture conditions on the proliferative life span of human fibroblasts isolated from different tissues and donors of different ages. Exp. Cell Res. 274, 275 /287. Butler, M.G., Tilburt, J., DeVries, A., Muralidhar, B., Aue, G., Hedges, L., Atkinson, J., Schwartz, H, 1998. Comparison of chromosome telomere integrity in multiple tissues from subjects at different ages. Cancer Genet. Cytogenet. 105, 138 /144. Cristofalo, V.J., Allen, R.G., Pgnolo, R.J., Martin, B.G., Beck, J.C., 1998. Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation. Proc. Natl. Acad. Sci. USA 95, 10614 /10619. de Lange, T., Shiue, L., Myers, R.M., Cox, D.R., Naylor, S.L., Killery, A.M., Varmus, H.E., 1990. Structure and variability of human chromosome ends. Mol. Cell. Biol. 10, 518 /527. Harley, C.B., Futcher, B., Greider, C.W., 1990. Telomeres shorten during aging of human fibroblasts. Nature 345, 458 /460. Hastie, N.D., Dempster, M., Dunlop, M.G., Thompson, A.M., Green, D.K., Allshire, R.C., 1990. Telomere reduction in human colorectal carcinoma and with ageing. Nature 346, 866 /868. Kang, M.K., Guo, W., Park, N.-H., 1998. Replicative senescence of normal human oral keratinocytes is associated with the loss of telomerase activity without shortening of telomeres. Cell Growth Differ. 9, 85 /95. Kang, M.K., Swee, J., Kim, R.H., Baluda, M.A., Park, N.-H., 2002. The telomeric length and heterogeneity decrease by age in normal human oral keratinocytes. Mech. Ageing Dev. 123, 585 /592. Karlseder, J., Smogorzewska, A., de Lange, T., 2002. Senescence induced by altered telomere state, not telomere loss. Science 295, 2446 /2449. Lucas, R.B., 1977. The epidemiology of oral tumors. Int. Dent. J. 27, 294 /298. Mondello, C., Petropoulou, C., Monti, D., Gonos, E.S., Franceschi, C., Nuzzo, F., 1999. Telomere length in fibroblasts and blood cells from healthy centenarians. Exp. Cell Res. 248, 234 /242. Tresini, M., Pignolo, R.J., Allen, R.G., Cristofalo, V.J., 1999. Effects of donor age on the expression of a marker of replicative senescence (EPC-1) in human dermal fibroblasts. Cell Physiol. 197, 11 /17. Vaziri, H., Scha¨chter, F., Uchida, I., Wei, L., Zhu, X., Effros, R., Cohen, D., Harley, C.B., 1993. Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am. J. Hum. Genet. 52, 661 /667.