- Email: [email protected]
Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells
Mechanisms of Ageing and Development 122 (2001) 1685– 1694 www.elsevier.com/locate/mechagedev
Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells Lianqing Yang a, Tetsuya Suwa a, Woodring E. Wright b, Jerry W. Shay b, Peter J. Hornsby a,* a
Huffington Center on Aging and Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza M320, Houston, TX 77030, USA b Department of Cell Biology, Uni6ersity of Texas Southwestern Medical Center, Dallas, TX 75390, USA Received 21 February 2001; received in revised form 16 April 2001; accepted 30 April 2001
Abstract Telomere shortening is the cause of replicative senescence of mammalian cells in culture and may be a cause of cellular aging in vivo. Some tissues clearly show telomere shortening during aging in humans, but the relationship between replication history and telomere length is obscured by complex relationships between stem cells and more differentiated cell types. Previous experiments on the adrenal cortex and human adrenocortical cells in culture indicate that the proliferative biology of this tissue is relatively simple; cell division occurs continuously throughout life, without evidence for a distinct stem cell compartment. In this tissue we investigated the relationship between telomere biology and replicative senescence by measuring replicative capacity and telomere length as a function of donor age. Cells cultured from adrenal tissue from donors of different ages showed a strong age-related decline in total replicative capacity, falling from about 50 population doublings for fetal cells to an almost total lack of division in culture for cells from older donors. Telomere restriction fragment (TRF) length was analyzed in the same sets of cells and decreased from a value of about 12 kb in fetal cells to approximately 7 kb in cells from older donors. The latter value is consistent with that in fibroblasts which have reached replicative senescence. Furthermore, there was a good correlation in individual donor samples between TRF length and -replicative capacity in culture. To confirm the relationship between telomere length, telom-
* Corresponding author. Tel.: +1-713-798-7334; fax: +1-713-798-6688. E-mail address: [email protected] (P.J. Hornsby). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 2 8 0 - 9
1686
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
erase, and replicative capacity, we measured telomere length in cells before and after infection with a retrovirus encoding hTERT, the catalytic component of human telomerase. The adult adrenal cortex does not have telomerase activity; cells after transduction with the hTERT retrovirus had high telomerase activity. Whereas control cells underwent a replication-dependent shortening in telomeres during long-term growth in culture, hTERT-modified cells maintained telomere length and are probably immortalized. Symmetric cell division in human adrenocortical cells, occurring slowly over the life span, is associated with progressive telomere shortening and may result in proliferative defects in vivo in old age, which could partly account for the age-related changes in the structure and function of the human adrenal cortex. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Adrenal cortex; Telomeres; Replicative senescence; Immortalization
1. Introduction Replicative senescence in culture results from progressive shortening of telomeres, which eventually triggers the operation of a cell cycle checkpoint, placing the cell in a state of irreversible growth arrest (Holt et al., 1996). The block to proliferation in replicative senescence is similar to that caused by double strand breaks in cellular DNA (Harley, 1991). Although the precise mechanism is still unclear, shortened telomeres appear to provide a DNA damage signal (Ouellette et al., 2000). Further cell division is then blocked by inhibitors of cell proliferation such as p21SDI1/WAF1/CIP1 and p16INK4A (Noda et al., 1994; Smith and Pereira-Smith, 1996). When this checkpoint is abrogated by oncoproteins such as SV40 T antigen, this first checkpoint (M1) is bypassed and cells eventually enter a second state, termed crisis or M2 (Holt et al., 1996). In this state the much shorter telomeres undergo end-to-end fusions, resulting in chromosomal breakage–fusion cycles that cause the cells to undergo apoptosis. There is massive loss of cells from the culture, whereas in replicative senescence (M1) cells do not die but enter a permanently nonreplicating state (Smith and Pereira-Smith, 1996). The finding that restoration of telomerase activity in cells that are normally telomerase negative both prevents shortening of telomeres and also immortalizes the cells shows that telomere shortening is the principal mechanism for replicative senescence (Bodnar et al., 1998). While these phenomena have been clearly described in cells in culture, their implications for tissues in vivo remain to be resolved. Proliferative defects in tissues during aging could result from telomere shortening, which has been observed in a variety of human tissues as a function of donor age (Hornsby, 2001). In many tissues the question is complicated by the existence of different compartments of cells; differentiated functions are performed by nondividing cells whereas cell division is performed by a stem cell compartment. However, some organs, like the liver and the adrenal cortex, have extensive regenerative potential, which results from the capacity of the majority of the cells both to perform differentiated functions and to divide (Hornsby, 1985; Alison, 1998). In the adrenal cortex most cell division occurs in the outer region, in the zona glomerulosa and outer zona
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1687
fasciculata, but there is no evidence that this is a stem cell zone or that cells in the rest of the cortex cannot divide. The kinetics of cell proliferation in the adrenal cortex in vivo are compatible with an extensive capacity for self-renewal of most cells in the tissue (Hornsby, 1985). Experiments involving transplantation of bovine adrenocortical cell clones in scid mice showed that 25% of unselected newly derived clones were able to form functional tissue in the host animals (Thomas et al., 1997). Cells giving rise to these clones are likely to be typical differentiated adrenocortical cells. Almost all cells in primary bovine adrenocortical cell cultures express differentiated features yet are also capable of DNA synthesis (Hornsby et al., 1992). Even though cell division rates are relatively low in the adrenal cortex in vivo (Hornsby, 1985), if cell division proceeds symmetrically then progressive exhaustion of replicative capacity can be expected, if the cells do not have sufficient telomerase activity to maintain telomere length. For these reasons the adrenal cortex provides an excellent opportunity to investigate the relationship of shortening of telomeres occurring during aging and replicative potential of the cells in culture. In the present experiments we measured telomere length and replicative capacity in cultures of adrenocortical cells isolated from human donors of different ages. 2. Methods
2.1. Cell and tissue preparation Human adrenal glands were obtained from kidney organ donors or from patients undergoing resection of the kidney for renal neoplasms. Organ donors were in the age range 0– 60 years and renal neoplasm patients were in the age range 50– 80 years. We are grateful to the staff of Lifegift, Houston, for their assistance in providing adrenals from kidney transplants. Fetal tissue samples were obtained from the University of Washington Laboratory for Embryology. Tissue fragments were dissociated to cell suspensions using enzymatic and mechanical dispersal (3 h incubation with 1 mg/ml type I-A collagenase and 0.1 mg/ml DNAse, both from Sigma, St. Louis, MO) (Hornsby and McAllister, 1991). Cell suspensions comprise about 90% adrenocortical cells; the viable cells in the population are \ 95% adrenocortical cells (McAllister and Hornsby, 1987). Primary cell suspensions were stored frozen in liquid nitrogen and were later thawed for telomere and telomerase measurements. For replicative potential measurements, cells were plated in Dulbecco’s Eagle’s medium/Ham’s F-12 1:1 with 10% fetal bovine serum, 10% heat-inactivated horse serum and 0.1 ng/ml recombinant basic FGF. This was supplemented with 1% v/v UltroSer G (Biosepra, Villeneuve-la-Garenne, France), a mixture of growth factors, as previously described. The gas phase used was 90% N2, 5% O2 and 5% CO2. Cells were grown in collagen I-coated tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ) and were subcultured at appropriate intervals using trypsin with a 1:5 split ratio. Cumulative population doublings were calculated as previously described (Hornsby et al., 1986).
1688
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
Human adrenocortical cells overexpressing hTERT were derived as described in detail elsewhere (Suwa et al., 2001).
2.2. Telomere restriction fragment analysis Mean telomere length was evaluated by using telomere restriction fragment (TRF) analysis, a variation of standard Southern blotting. DNAs isolated from different cell preparations were digested with a mixture of restriction enzymes and resolved on a 0.7% agarose gel (Ouellette et al., 2000). The dried gel was hybridized with a 32P-labeled oligonucleotide, (TTAGGG)3, exposed to a Phosphorimager screen, and TRFs were calculated as described in detail elsewhere (Ouellette et al., 2000) using two or three separate measurements for each sample.
2.3. Measurement of telomerase acti6ity by telomerase repeat amplification protocol (TRAP) assay Cell pellets were extracted with 0.5 ml ice-cold CHAPS lysis buffer (10 mM Tris– HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM b-mercaptoethanol, 10% glycerol, and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) on ice for 30 min (Kim et al., 1994). Cell lysates were clarified by centrifugation at 14 000×g for 30 min at 4 °C. The TRAP assay was performed on the supernatants using the TRAP-EZE kit (Intergen, Purchase, NY). Cell extract (2 ml) was mixed with 5 ml of 10 × TRAP reaction buffer (200 mM Tris– HCl, pH 8.3, 15 mM MgCl2, 630 mM KCl, 0.5% Tween 20, and 10 mM EGTA), 1 ml 50× modified dNTP mix, 2 ml TS primer, 1 ml modified primer mix, 0.4 ml (4 mCi) [h-32P]dCTP, 0.4 ml Taq polymerase (ExTaq, Takara, Kyoto, Japan) and 38.2 ml water. The incubation was performed at 37 °C for 30 min. PCR was then performed (94 °C/30 s, 59 °C/30 s for 29 cycles). Products (20 ml) were separated by 10% PAGE. Gels were dried and exposed to X-ray film for appropriate lengths of time.
3. Results The replicative capacity of human adrenocortical cells in culture was determined using cells prepared from tissue samples from donors of various ages. Donor tissue specimens included some derived from fetal sources (20–24 weeks gestational age) and others in a postnatal age range from 8 months to 76 years. Cells were allowed to proliferate and were subcultured as necessary until growth ceased, defined as the failure of the cells to regrow to confluence within a period of 2 weeks following subculture. The total number of population doublings achieved was then calculated (Fig. 1). It was not possible to obtain accurate information on the replicative capacity of adrenocortical cells obtained from donors in the range 40–80 years of age. Although adrenocortical cells from donors in this age range attached firmly to culture dishes, appeared healthy, and secreted steroids, very little proliferation of
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1689
these cells was observed. As previously reported (McAllister and Hornsby, 1987), there are a small number of non-adrenocortical cells (B 5%) in the starting cell population; when adrenocortical cells do not divide at an adequate rate, the non-adrenocortical cell population eventually overgrows the culture. We concluded that the cells that proliferated in culture from older donors are not adrenocortical cells because: (a) proliferation was focal, (b) proliferating cells did not resemble adrenocortical cells, and (c) they did not show the rounding response observed in adrenocortical cells when they are incubated with ACTH or cyclic AMP (McAllister and Hornsby, 1987). Although it is likely that the population of adrenocortical cells from these older donors may have been able to divide a few times, overgrowth by non-adrenocortical cells prevented us from designating a specific number. We measured telomere length in the starting cell populations used to prepare these cultures. Southern blotting was performed on DNA digested to produce telomere restriction fragments (TRFs) (Fig. 2a). TRF length decreased from 11– 12 kb to ca. 7 kb over the range from fetal to age 78. The data fit a linear decline with a rate of decrease of ca. 4496 bp/year with r 2 = 0.6 (Fig. 2b). When TRF length was plotted against replicative capacity for the various cell populations, a strong dependence of future replicative capacity on initial telomere length was observed (Fig. 2c).
Fig. 1. Replicative capacity of human adrenocortical cells in culture as a function of donor age. Cells were prepared from adrenocortical tissue from donors of the indicated ages (F, fetal tissue samples). Cells were placed in culture, as described in Section 2, and were allowed to divide, with subculturing as necessary, until cell division ceased. The total number of population doublings (PD) achieved is plotted ( ). In cultures derived from donors in the range of 40 to 80 years of age most adrenocortical cells did not divide significantly in culture and under these circumstances non-adrenocortical cells in the culture eventually overgrew the adrenocortical cells (see text for more details). Cultures for which this phenomenon was observed are shown as (organ donors) and (patients with renal neoplasms).
1690
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
Fig. 2. Telomere restriction fragment length as a function of age in human adrenocortical cells. (a) Southern blot showing telomere restriction fragments in DNA from cells from various ages of donors. DNA was prepared from samples of cells dispersed from adrenocortical tissue obtained from donors of the indicated ages. The first two samples are from fetal tissue of the indicated gestational age. DNA was digested with enzymes, electrophoresed on agarose, blotted and hybridized to a telomeric probe as described in Methods. TRFs were calculated from Phosphorimager data and are indicated below each lane. (b) TRFs from a larger set of samples are plotted and the best fit line is shown. (c) TRF is plotted as a function of replicative capacity, for those tissue samples for which both measurements were available.
Additional data were obtained to confirm the relationship of shortened telomeres and replicative potential observed in Figs. 1 and 2. It is predicted that, if telomere length is the major determinant of replicative senescence in this cell type, overexpression of hTERT should cause cells to bypass the senescence (M1) point in culture while telomere length is maintained at a value above that associated with senescence. Fig. 3 shows that freshly isolated human adrenocortical cells have undetectable telomerase activity; therefore presumably the adrenal cortex as a tissue lacks telomerase activity. However, as we have shown elsewhere (Suwa et al., 2001), telomerase activity may be detected weakly in primary cultures of human adrenocortical cells when a large number of cells are dividing, yet not in long-term cultures, despite continued cell division. Cultured adrenocortical cells overexpressing hTERT after infection with an hTERT-encoding retrovirus showed an extended replicative potential (currently \ 75 PD, in contrast to a total replicative potential of 23 PD in the untreated cells) and also showed a TRF value (9.6 kb at PDL 35) that was greater than that in the untreated cells (7.9 kb at PDL 20).
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1691
4. Discussion The data on telomere length in human adrenocortical cells presented here show that telomere length declines throughout life and that the decrease correlates with a marked decline in total replicative potential in culture. Shortened telomeres are the likely cause of the lack of proliferation in cells isolated from older donors, because: (a) the telomere length in cells from older donors is close to the M1 length (7 kb; Allsopp et al., (1992)), and (b) overexpression of hTERT in cultured adrenocortical cells enables extended proliferation of cells accompanied by increased telomere length. The present experiments focus on data associating replicative potential and telomere length as a function of donor age, and its implication for aging of the adrenal cortex. Another study (Suwa et al., 2001) presents data on telomerase activity in cultured adrenocortical cells; in the present study we confirm that hTERT overexpression has the predicted effect if replicative potential is primarily determined by telomere length. hTERT-modified cells exhibit an extended replicative potential and are probably immortalized. Over 30 years ago it was observed that the replicative capacity of human fibroblasts in culture decreases as a function of donor age (Martin et al., 1970), although there was much variation within each decade of age in the maximal and minimal proliferative capacity of the different cell samples. Subsequently the generality of the observation was challenged by finding that the decrease as a
Fig. 3. Telomerase activity and effects of telomerase in human adrenocortical cells. (a) Telomerase repeat amplification protocol (TRAP) assay performed on human adrenocortical cells. Extracts were made from two adrenocortical cell preparations (both from 28-year-old males) and from human adrenocortical cells with ectopic expression of hTERT following retroviral infection. Buffer-only and 3T3 cell extract lanes were included as negative and positive controls, respectively. (b) Southern blot performed as described in Fig. 2 showing effect on telomere length of hTERT expression. DNA was prepared from control cells at PDL 20 and from hTERT-expressing cells at PDL 35 (see text for details).
1692
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
function of donor age applied only to fibroblasts isolated from diabetic and ‘prediabetic’ patients (Goldstein et al., 1978), and was not evident in fibroblasts obtained from non-sun exposed skin (Gilchrest, 1980; Cristofalo et al., 1998). A significant problem with these studies is that fibroblasts in tissues in vivo are probably mostly in a nonreplicating state over the life span of an individual, and tissues probably contain cells with heterogeneous prior proliferative histories (Hornsby, 2001). Therefore a random biopsy may comprise a mixture of proliferatively young and old cells. Since it takes only three divisions for young cells to take over the population if 12.5% of the population is young (12.5 25 50100%), measurements of the entire population will show an altered cultured life span only if the fraction of ‘reserve’ young cells is low. Since proliferation in the dermis probably occurs primarily in response to damage, it is not at all surprising that protected skin of the inner arm would have a lower proliferative history than that from the outer arm. Fibroblasts from the dermis of the outer arm do show declines in both telomere length and proliferative capacity as a function of donor age (Allsopp et al., 1992). Additionally, cells from patients with Werner syndrome, a segmental progeroid syndrome, have a decreased replicative potential and accelerated telomere shortening (Salk, 1985; Schulz et al., 1996). Fibroblasts are readily cultured and most cell culture data on the molecular basis for replicative senescence have come from studies on fibroblasts. However, methods for isolating fibroblasts are hard to standardize, the cells are heterogeneous, and the properties of the cells are highly dependent on the precise site of the biopsy. It is desirable to study the effects of donor age on replicative capacity in cells which can be reproducibly isolated as pure populations and which show some degree of proliferation throughout life. Studies on non-fibroblast cells have often shown striking decreases in proliferative capacity (Hornsby, 2001). For example, proliferative capacity is closely related to telomere length in endothelial cells. Telomere length in endothelial cells decreased as a function of donor age, with a greater decline being observed in cells isolated from the iliac artery in comparison to cells from the thoracic artery (Chang and Harley, 1995). The greater decline in telomere length in the iliac artery endothelial cells is thought to reflect an increased proliferation in response to chronic damage to the endothelium due to increased turbulence in the iliac versus thoracic artery. The present studies add to these previous observations by demonstrating the relationships among donor age, telomere length and replicative potential in the adrenal cortex. This is a tissue that does not have a known stem cell compartment, yet has an extensive regenerative capacity that arises from the potential of adrenocortical cells both to perform differentiated functions and to divide. Whether telomere shortening in vivo actually results in cells that can no longer divide in situ is not directly determined by these experiments. However, the progressive atrophy and lowered cell division noted from clinical studies of the human adrenal cortex is consistent with loss of the ability of the cells to divide in vivo (Neville and O’Hare, 1982). Although detailed in vivo measurements are lacking, the loss of telomeric DNA at 41 bp/year would require only that cells divide on average once per year, and also that cell division occurs symmetrically, not asymmetrically as in a stem cell
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1693
compartment. The decreased width of the cortex during aging is associated with a loss of the zona reticularis, the inner zone of the cortex that secretes dehydroepiandrosterone (Parker et al., 1997). The present data suggest a model in which the decline in telomere length resulting from proliferation of the cells over the life span eventually causes slowing or failure of further proliferation. Together with death of adrenocortical cells over the life span, this eventually results in a thinner cortex that is unable to maintain the inner zone, with a consequent decline in blood levels of DHEA (Hornsby, 1995). Moreover, hyperplasias (nodules and adenomas) that develop in the adrenal cortex during aging (Neville and O’Hare, 1982) might also result from loss of the ability of the majority of cells to divide. The consequent increased growth stimulus acting on the minority of cells capable of cell division may produce multiple focal growths. These experiments are consistent with the concept that cell proliferation occurring over the human life span causes telomere shortening in the adrenal cortex. Cultures from older donors are then initiated with cells that have shorter telomeres and thus exhibit a lowered remaining proliferative potential, because continued cell division in culture shortens telomeres to a point that produces replicative senescence. Telomere shortening in the human adrenal cortex may be partly responsible for age-related changes in adrenocortical structure and declines in function. Acknowledgements This work was supported by grants from the National Institute on Aging (AG12287 and AG13663 to PJH and AG01228 to WEW). References Alison, M., 1998. Liver stem cells: a two compartment system. Curr. Opin. Cell Biol. 10, 710 – 715. Allsopp, R.C., Vaziri, H., Patterson, C., et al., 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89, 10114 – 10118. Bodnar, A.G., Ouellette, M., Frolkis, M., et al., 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349 – 352. Chang, E., Harley, C.B., 1995. Telomere length and replicative aging in human vascular tissues. Proc. Natl. Acad. Sci. USA 92, 11190 –11194. Cristofalo, V.J., Allen, R.G., Pignolo, 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. Gilchrest, B.A., 1980. Prior chronic sun exposure decreases the lifespan of human skin fibroblasts in vitro. J. Gerontol. 35, 537 –541. Goldstein, S., Moerman, E.J., Soeldner, J.S., Gleason, R.E., Barnett, D.M., 1978. Chronologic and physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic, and normal donors. Science 199, 781 –782. Harley, C.B., 1991. Telomere loss: mitotic clock or genetic time bomb? Mutat. Res. 256, 271 – 282. Holt, S.E., Shay, J.W., Wright, W.E., 1996. Refining the telomere – telomerase hypothesis of aging and cancer. Nat. Biotechnol. 14, 836 –839. Hornsby, P.J., 1985. The regulation of adrenocortical function by control of growth and structure. In: Anderson, D.C., Winter, J.S.D. (Eds.), Adrenal Cortex. Butterworth, London, pp. 1 – 31.
1694
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
Hornsby, P.J., 1995. Biosynthesis of DHEAS by the human adrenal cortex and its age-related decline. Ann. NY Acad. Sci. 774, 29 –46. Hornsby, P.J., 2001. Cell proliferation in mammalian aging. In: Masoro, E.J., Austad, S.N. (Eds.), Handbook of the Biology of Aging, 5th edn. Academic Press, San Diego, CA, pp. 207 – 245. Hornsby, P.J., McAllister, J.M., 1991. Culturing steroidogenic cells. In: Waterman, M.R., Johnson, E.F. (Eds.), Methods in Enzymology, vol. 206. Academic Press, San Diego, CA, pp. 371 – 380. Hornsby, P.J., Aldern, K.A., Harris, S.E., 1986. Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence. J. Cell. Physiol. 129, 395 – 402. Hornsby, P.J., Cheng, C.Y., Lala, D.S., Maghsoudlou, S.S., Raju, S.G., Yang, L., 1992. Changes in gene expression during senescence of adrenocortical cells in culture. J. Steroid Biochem. Mol. Biol. 43, 385–395. Kim, N.W., Piatyszek, M.A., Prowse, K.R., et al., 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011 – 2015. Martin, G.M., Sprague, C.A., Epstein, C.A., 1970. Replicative life-span of cultivated human cells: effects of donor’s age, tissue and genotype. Lab. Invest. 23, 86 – 92. McAllister, J.M., Hornsby, P.J., 1987. Improved clonal and non-clonal growth of human, rat, and bovine adrenocortical cells in culture. In Vitro Cell. Dev. Biol. 23, 677 – 685. Neville, A.M., O’Hare, M.J., 1982. The Human Adrenal Cortex. Pathology and Biology —an Integrated Approach. Springer, Berlin. Noda, A., Ning, Y., Venable, S.F., Pereira-Smith, O.M., Smith, J.R., 1994. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res. 211, 90 – 98. Ouellette, M.M., Liao, M., Herbert, B.S., et al., 2000. Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J. Biol. Chem. 275, 10072 – 10076. Parker, C.R., Mixon, R.L., Brissie, R.M., Grizzle, W.E., 1997. Aging alters zonation in the adrenal cortex of men. J. Clin. Endocrinol. Metab. 82, 3898 – 3901. Salk, D., 1985. Werner syndrome: a review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations. In: Salk, D., Fujiwara, Y., Martin, G.M. (Eds.), Werner’s Syndrome and Human Aging. Plenum, New York, pp. 121 – 160. Schulz, V.P., Zakian, V.A., Ogburn, C.E., et al., 1996. Accelerated loss of telomeric repeats may not explain accelerated replicative decline of Werner syndrome cells. Hum. Genet. 97, 750 – 754. Smith, J.R., Pereira-Smith, O.M., 1996. Replicative senescence: implications for in vivo aging and tumor suppression. Science 273, 63 –67. Suwa, T., Yang, L., Hornsby, P.J., 2001. Telomerase activity in primary cultures of bovine and human adrenocortical cells. J. Endocrinol., in press. Thomas, M., Northrup, S.R., Hornsby, P.J., 1997. Adrenocortical tissue formed by transplantation of normal clones of bovine adrenocortical cells in scid mice replaces the essential functions of the animals’ adrenal glands. Nat. Med. 3, 978 – 983.
Telomere shortening and decline in replicative potential as a function of donor age in human adrenocortical cells Lianqing Yang a, Tetsuya Suwa a, Woodring E. Wright b, Jerry W. Shay b, Peter J. Hornsby a,* a
Huffington Center on Aging and Department of Molecular and Cellular Biology, Baylor College of Medicine, 1 Baylor Plaza M320, Houston, TX 77030, USA b Department of Cell Biology, Uni6ersity of Texas Southwestern Medical Center, Dallas, TX 75390, USA Received 21 February 2001; received in revised form 16 April 2001; accepted 30 April 2001
Abstract Telomere shortening is the cause of replicative senescence of mammalian cells in culture and may be a cause of cellular aging in vivo. Some tissues clearly show telomere shortening during aging in humans, but the relationship between replication history and telomere length is obscured by complex relationships between stem cells and more differentiated cell types. Previous experiments on the adrenal cortex and human adrenocortical cells in culture indicate that the proliferative biology of this tissue is relatively simple; cell division occurs continuously throughout life, without evidence for a distinct stem cell compartment. In this tissue we investigated the relationship between telomere biology and replicative senescence by measuring replicative capacity and telomere length as a function of donor age. Cells cultured from adrenal tissue from donors of different ages showed a strong age-related decline in total replicative capacity, falling from about 50 population doublings for fetal cells to an almost total lack of division in culture for cells from older donors. Telomere restriction fragment (TRF) length was analyzed in the same sets of cells and decreased from a value of about 12 kb in fetal cells to approximately 7 kb in cells from older donors. The latter value is consistent with that in fibroblasts which have reached replicative senescence. Furthermore, there was a good correlation in individual donor samples between TRF length and -replicative capacity in culture. To confirm the relationship between telomere length, telom-
* Corresponding author. Tel.: +1-713-798-7334; fax: +1-713-798-6688. E-mail address: [email protected] (P.J. Hornsby). 0047-6374/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 1 ) 0 0 2 8 0 - 9
1686
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
erase, and replicative capacity, we measured telomere length in cells before and after infection with a retrovirus encoding hTERT, the catalytic component of human telomerase. The adult adrenal cortex does not have telomerase activity; cells after transduction with the hTERT retrovirus had high telomerase activity. Whereas control cells underwent a replication-dependent shortening in telomeres during long-term growth in culture, hTERT-modified cells maintained telomere length and are probably immortalized. Symmetric cell division in human adrenocortical cells, occurring slowly over the life span, is associated with progressive telomere shortening and may result in proliferative defects in vivo in old age, which could partly account for the age-related changes in the structure and function of the human adrenal cortex. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Adrenal cortex; Telomeres; Replicative senescence; Immortalization
1. Introduction Replicative senescence in culture results from progressive shortening of telomeres, which eventually triggers the operation of a cell cycle checkpoint, placing the cell in a state of irreversible growth arrest (Holt et al., 1996). The block to proliferation in replicative senescence is similar to that caused by double strand breaks in cellular DNA (Harley, 1991). Although the precise mechanism is still unclear, shortened telomeres appear to provide a DNA damage signal (Ouellette et al., 2000). Further cell division is then blocked by inhibitors of cell proliferation such as p21SDI1/WAF1/CIP1 and p16INK4A (Noda et al., 1994; Smith and Pereira-Smith, 1996). When this checkpoint is abrogated by oncoproteins such as SV40 T antigen, this first checkpoint (M1) is bypassed and cells eventually enter a second state, termed crisis or M2 (Holt et al., 1996). In this state the much shorter telomeres undergo end-to-end fusions, resulting in chromosomal breakage–fusion cycles that cause the cells to undergo apoptosis. There is massive loss of cells from the culture, whereas in replicative senescence (M1) cells do not die but enter a permanently nonreplicating state (Smith and Pereira-Smith, 1996). The finding that restoration of telomerase activity in cells that are normally telomerase negative both prevents shortening of telomeres and also immortalizes the cells shows that telomere shortening is the principal mechanism for replicative senescence (Bodnar et al., 1998). While these phenomena have been clearly described in cells in culture, their implications for tissues in vivo remain to be resolved. Proliferative defects in tissues during aging could result from telomere shortening, which has been observed in a variety of human tissues as a function of donor age (Hornsby, 2001). In many tissues the question is complicated by the existence of different compartments of cells; differentiated functions are performed by nondividing cells whereas cell division is performed by a stem cell compartment. However, some organs, like the liver and the adrenal cortex, have extensive regenerative potential, which results from the capacity of the majority of the cells both to perform differentiated functions and to divide (Hornsby, 1985; Alison, 1998). In the adrenal cortex most cell division occurs in the outer region, in the zona glomerulosa and outer zona
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1687
fasciculata, but there is no evidence that this is a stem cell zone or that cells in the rest of the cortex cannot divide. The kinetics of cell proliferation in the adrenal cortex in vivo are compatible with an extensive capacity for self-renewal of most cells in the tissue (Hornsby, 1985). Experiments involving transplantation of bovine adrenocortical cell clones in scid mice showed that 25% of unselected newly derived clones were able to form functional tissue in the host animals (Thomas et al., 1997). Cells giving rise to these clones are likely to be typical differentiated adrenocortical cells. Almost all cells in primary bovine adrenocortical cell cultures express differentiated features yet are also capable of DNA synthesis (Hornsby et al., 1992). Even though cell division rates are relatively low in the adrenal cortex in vivo (Hornsby, 1985), if cell division proceeds symmetrically then progressive exhaustion of replicative capacity can be expected, if the cells do not have sufficient telomerase activity to maintain telomere length. For these reasons the adrenal cortex provides an excellent opportunity to investigate the relationship of shortening of telomeres occurring during aging and replicative potential of the cells in culture. In the present experiments we measured telomere length and replicative capacity in cultures of adrenocortical cells isolated from human donors of different ages. 2. Methods
2.1. Cell and tissue preparation Human adrenal glands were obtained from kidney organ donors or from patients undergoing resection of the kidney for renal neoplasms. Organ donors were in the age range 0– 60 years and renal neoplasm patients were in the age range 50– 80 years. We are grateful to the staff of Lifegift, Houston, for their assistance in providing adrenals from kidney transplants. Fetal tissue samples were obtained from the University of Washington Laboratory for Embryology. Tissue fragments were dissociated to cell suspensions using enzymatic and mechanical dispersal (3 h incubation with 1 mg/ml type I-A collagenase and 0.1 mg/ml DNAse, both from Sigma, St. Louis, MO) (Hornsby and McAllister, 1991). Cell suspensions comprise about 90% adrenocortical cells; the viable cells in the population are \ 95% adrenocortical cells (McAllister and Hornsby, 1987). Primary cell suspensions were stored frozen in liquid nitrogen and were later thawed for telomere and telomerase measurements. For replicative potential measurements, cells were plated in Dulbecco’s Eagle’s medium/Ham’s F-12 1:1 with 10% fetal bovine serum, 10% heat-inactivated horse serum and 0.1 ng/ml recombinant basic FGF. This was supplemented with 1% v/v UltroSer G (Biosepra, Villeneuve-la-Garenne, France), a mixture of growth factors, as previously described. The gas phase used was 90% N2, 5% O2 and 5% CO2. Cells were grown in collagen I-coated tissue culture dishes (Becton Dickinson, Franklin Lakes, NJ) and were subcultured at appropriate intervals using trypsin with a 1:5 split ratio. Cumulative population doublings were calculated as previously described (Hornsby et al., 1986).
1688
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
Human adrenocortical cells overexpressing hTERT were derived as described in detail elsewhere (Suwa et al., 2001).
2.2. Telomere restriction fragment analysis Mean telomere length was evaluated by using telomere restriction fragment (TRF) analysis, a variation of standard Southern blotting. DNAs isolated from different cell preparations were digested with a mixture of restriction enzymes and resolved on a 0.7% agarose gel (Ouellette et al., 2000). The dried gel was hybridized with a 32P-labeled oligonucleotide, (TTAGGG)3, exposed to a Phosphorimager screen, and TRFs were calculated as described in detail elsewhere (Ouellette et al., 2000) using two or three separate measurements for each sample.
2.3. Measurement of telomerase acti6ity by telomerase repeat amplification protocol (TRAP) assay Cell pellets were extracted with 0.5 ml ice-cold CHAPS lysis buffer (10 mM Tris– HCl, pH 7.5, 1 mM MgCl2, 1 mM EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM b-mercaptoethanol, 10% glycerol, and 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) on ice for 30 min (Kim et al., 1994). Cell lysates were clarified by centrifugation at 14 000×g for 30 min at 4 °C. The TRAP assay was performed on the supernatants using the TRAP-EZE kit (Intergen, Purchase, NY). Cell extract (2 ml) was mixed with 5 ml of 10 × TRAP reaction buffer (200 mM Tris– HCl, pH 8.3, 15 mM MgCl2, 630 mM KCl, 0.5% Tween 20, and 10 mM EGTA), 1 ml 50× modified dNTP mix, 2 ml TS primer, 1 ml modified primer mix, 0.4 ml (4 mCi) [h-32P]dCTP, 0.4 ml Taq polymerase (ExTaq, Takara, Kyoto, Japan) and 38.2 ml water. The incubation was performed at 37 °C for 30 min. PCR was then performed (94 °C/30 s, 59 °C/30 s for 29 cycles). Products (20 ml) were separated by 10% PAGE. Gels were dried and exposed to X-ray film for appropriate lengths of time.
3. Results The replicative capacity of human adrenocortical cells in culture was determined using cells prepared from tissue samples from donors of various ages. Donor tissue specimens included some derived from fetal sources (20–24 weeks gestational age) and others in a postnatal age range from 8 months to 76 years. Cells were allowed to proliferate and were subcultured as necessary until growth ceased, defined as the failure of the cells to regrow to confluence within a period of 2 weeks following subculture. The total number of population doublings achieved was then calculated (Fig. 1). It was not possible to obtain accurate information on the replicative capacity of adrenocortical cells obtained from donors in the range 40–80 years of age. Although adrenocortical cells from donors in this age range attached firmly to culture dishes, appeared healthy, and secreted steroids, very little proliferation of
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1689
these cells was observed. As previously reported (McAllister and Hornsby, 1987), there are a small number of non-adrenocortical cells (B 5%) in the starting cell population; when adrenocortical cells do not divide at an adequate rate, the non-adrenocortical cell population eventually overgrows the culture. We concluded that the cells that proliferated in culture from older donors are not adrenocortical cells because: (a) proliferation was focal, (b) proliferating cells did not resemble adrenocortical cells, and (c) they did not show the rounding response observed in adrenocortical cells when they are incubated with ACTH or cyclic AMP (McAllister and Hornsby, 1987). Although it is likely that the population of adrenocortical cells from these older donors may have been able to divide a few times, overgrowth by non-adrenocortical cells prevented us from designating a specific number. We measured telomere length in the starting cell populations used to prepare these cultures. Southern blotting was performed on DNA digested to produce telomere restriction fragments (TRFs) (Fig. 2a). TRF length decreased from 11– 12 kb to ca. 7 kb over the range from fetal to age 78. The data fit a linear decline with a rate of decrease of ca. 4496 bp/year with r 2 = 0.6 (Fig. 2b). When TRF length was plotted against replicative capacity for the various cell populations, a strong dependence of future replicative capacity on initial telomere length was observed (Fig. 2c).
Fig. 1. Replicative capacity of human adrenocortical cells in culture as a function of donor age. Cells were prepared from adrenocortical tissue from donors of the indicated ages (F, fetal tissue samples). Cells were placed in culture, as described in Section 2, and were allowed to divide, with subculturing as necessary, until cell division ceased. The total number of population doublings (PD) achieved is plotted ( ). In cultures derived from donors in the range of 40 to 80 years of age most adrenocortical cells did not divide significantly in culture and under these circumstances non-adrenocortical cells in the culture eventually overgrew the adrenocortical cells (see text for more details). Cultures for which this phenomenon was observed are shown as (organ donors) and (patients with renal neoplasms).
1690
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
Fig. 2. Telomere restriction fragment length as a function of age in human adrenocortical cells. (a) Southern blot showing telomere restriction fragments in DNA from cells from various ages of donors. DNA was prepared from samples of cells dispersed from adrenocortical tissue obtained from donors of the indicated ages. The first two samples are from fetal tissue of the indicated gestational age. DNA was digested with enzymes, electrophoresed on agarose, blotted and hybridized to a telomeric probe as described in Methods. TRFs were calculated from Phosphorimager data and are indicated below each lane. (b) TRFs from a larger set of samples are plotted and the best fit line is shown. (c) TRF is plotted as a function of replicative capacity, for those tissue samples for which both measurements were available.
Additional data were obtained to confirm the relationship of shortened telomeres and replicative potential observed in Figs. 1 and 2. It is predicted that, if telomere length is the major determinant of replicative senescence in this cell type, overexpression of hTERT should cause cells to bypass the senescence (M1) point in culture while telomere length is maintained at a value above that associated with senescence. Fig. 3 shows that freshly isolated human adrenocortical cells have undetectable telomerase activity; therefore presumably the adrenal cortex as a tissue lacks telomerase activity. However, as we have shown elsewhere (Suwa et al., 2001), telomerase activity may be detected weakly in primary cultures of human adrenocortical cells when a large number of cells are dividing, yet not in long-term cultures, despite continued cell division. Cultured adrenocortical cells overexpressing hTERT after infection with an hTERT-encoding retrovirus showed an extended replicative potential (currently \ 75 PD, in contrast to a total replicative potential of 23 PD in the untreated cells) and also showed a TRF value (9.6 kb at PDL 35) that was greater than that in the untreated cells (7.9 kb at PDL 20).
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1691
4. Discussion The data on telomere length in human adrenocortical cells presented here show that telomere length declines throughout life and that the decrease correlates with a marked decline in total replicative potential in culture. Shortened telomeres are the likely cause of the lack of proliferation in cells isolated from older donors, because: (a) the telomere length in cells from older donors is close to the M1 length (7 kb; Allsopp et al., (1992)), and (b) overexpression of hTERT in cultured adrenocortical cells enables extended proliferation of cells accompanied by increased telomere length. The present experiments focus on data associating replicative potential and telomere length as a function of donor age, and its implication for aging of the adrenal cortex. Another study (Suwa et al., 2001) presents data on telomerase activity in cultured adrenocortical cells; in the present study we confirm that hTERT overexpression has the predicted effect if replicative potential is primarily determined by telomere length. hTERT-modified cells exhibit an extended replicative potential and are probably immortalized. Over 30 years ago it was observed that the replicative capacity of human fibroblasts in culture decreases as a function of donor age (Martin et al., 1970), although there was much variation within each decade of age in the maximal and minimal proliferative capacity of the different cell samples. Subsequently the generality of the observation was challenged by finding that the decrease as a
Fig. 3. Telomerase activity and effects of telomerase in human adrenocortical cells. (a) Telomerase repeat amplification protocol (TRAP) assay performed on human adrenocortical cells. Extracts were made from two adrenocortical cell preparations (both from 28-year-old males) and from human adrenocortical cells with ectopic expression of hTERT following retroviral infection. Buffer-only and 3T3 cell extract lanes were included as negative and positive controls, respectively. (b) Southern blot performed as described in Fig. 2 showing effect on telomere length of hTERT expression. DNA was prepared from control cells at PDL 20 and from hTERT-expressing cells at PDL 35 (see text for details).
1692
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
function of donor age applied only to fibroblasts isolated from diabetic and ‘prediabetic’ patients (Goldstein et al., 1978), and was not evident in fibroblasts obtained from non-sun exposed skin (Gilchrest, 1980; Cristofalo et al., 1998). A significant problem with these studies is that fibroblasts in tissues in vivo are probably mostly in a nonreplicating state over the life span of an individual, and tissues probably contain cells with heterogeneous prior proliferative histories (Hornsby, 2001). Therefore a random biopsy may comprise a mixture of proliferatively young and old cells. Since it takes only three divisions for young cells to take over the population if 12.5% of the population is young (12.5 25 50100%), measurements of the entire population will show an altered cultured life span only if the fraction of ‘reserve’ young cells is low. Since proliferation in the dermis probably occurs primarily in response to damage, it is not at all surprising that protected skin of the inner arm would have a lower proliferative history than that from the outer arm. Fibroblasts from the dermis of the outer arm do show declines in both telomere length and proliferative capacity as a function of donor age (Allsopp et al., 1992). Additionally, cells from patients with Werner syndrome, a segmental progeroid syndrome, have a decreased replicative potential and accelerated telomere shortening (Salk, 1985; Schulz et al., 1996). Fibroblasts are readily cultured and most cell culture data on the molecular basis for replicative senescence have come from studies on fibroblasts. However, methods for isolating fibroblasts are hard to standardize, the cells are heterogeneous, and the properties of the cells are highly dependent on the precise site of the biopsy. It is desirable to study the effects of donor age on replicative capacity in cells which can be reproducibly isolated as pure populations and which show some degree of proliferation throughout life. Studies on non-fibroblast cells have often shown striking decreases in proliferative capacity (Hornsby, 2001). For example, proliferative capacity is closely related to telomere length in endothelial cells. Telomere length in endothelial cells decreased as a function of donor age, with a greater decline being observed in cells isolated from the iliac artery in comparison to cells from the thoracic artery (Chang and Harley, 1995). The greater decline in telomere length in the iliac artery endothelial cells is thought to reflect an increased proliferation in response to chronic damage to the endothelium due to increased turbulence in the iliac versus thoracic artery. The present studies add to these previous observations by demonstrating the relationships among donor age, telomere length and replicative potential in the adrenal cortex. This is a tissue that does not have a known stem cell compartment, yet has an extensive regenerative capacity that arises from the potential of adrenocortical cells both to perform differentiated functions and to divide. Whether telomere shortening in vivo actually results in cells that can no longer divide in situ is not directly determined by these experiments. However, the progressive atrophy and lowered cell division noted from clinical studies of the human adrenal cortex is consistent with loss of the ability of the cells to divide in vivo (Neville and O’Hare, 1982). Although detailed in vivo measurements are lacking, the loss of telomeric DNA at 41 bp/year would require only that cells divide on average once per year, and also that cell division occurs symmetrically, not asymmetrically as in a stem cell
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
1693
compartment. The decreased width of the cortex during aging is associated with a loss of the zona reticularis, the inner zone of the cortex that secretes dehydroepiandrosterone (Parker et al., 1997). The present data suggest a model in which the decline in telomere length resulting from proliferation of the cells over the life span eventually causes slowing or failure of further proliferation. Together with death of adrenocortical cells over the life span, this eventually results in a thinner cortex that is unable to maintain the inner zone, with a consequent decline in blood levels of DHEA (Hornsby, 1995). Moreover, hyperplasias (nodules and adenomas) that develop in the adrenal cortex during aging (Neville and O’Hare, 1982) might also result from loss of the ability of the majority of cells to divide. The consequent increased growth stimulus acting on the minority of cells capable of cell division may produce multiple focal growths. These experiments are consistent with the concept that cell proliferation occurring over the human life span causes telomere shortening in the adrenal cortex. Cultures from older donors are then initiated with cells that have shorter telomeres and thus exhibit a lowered remaining proliferative potential, because continued cell division in culture shortens telomeres to a point that produces replicative senescence. Telomere shortening in the human adrenal cortex may be partly responsible for age-related changes in adrenocortical structure and declines in function. Acknowledgements This work was supported by grants from the National Institute on Aging (AG12287 and AG13663 to PJH and AG01228 to WEW). References Alison, M., 1998. Liver stem cells: a two compartment system. Curr. Opin. Cell Biol. 10, 710 – 715. Allsopp, R.C., Vaziri, H., Patterson, C., et al., 1992. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89, 10114 – 10118. Bodnar, A.G., Ouellette, M., Frolkis, M., et al., 1998. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349 – 352. Chang, E., Harley, C.B., 1995. Telomere length and replicative aging in human vascular tissues. Proc. Natl. Acad. Sci. USA 92, 11190 –11194. Cristofalo, V.J., Allen, R.G., Pignolo, 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. Gilchrest, B.A., 1980. Prior chronic sun exposure decreases the lifespan of human skin fibroblasts in vitro. J. Gerontol. 35, 537 –541. Goldstein, S., Moerman, E.J., Soeldner, J.S., Gleason, R.E., Barnett, D.M., 1978. Chronologic and physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic, and normal donors. Science 199, 781 –782. Harley, C.B., 1991. Telomere loss: mitotic clock or genetic time bomb? Mutat. Res. 256, 271 – 282. Holt, S.E., Shay, J.W., Wright, W.E., 1996. Refining the telomere – telomerase hypothesis of aging and cancer. Nat. Biotechnol. 14, 836 –839. Hornsby, P.J., 1985. The regulation of adrenocortical function by control of growth and structure. In: Anderson, D.C., Winter, J.S.D. (Eds.), Adrenal Cortex. Butterworth, London, pp. 1 – 31.
1694
L. Yang et al. / Mechanisms of Ageing and De6elopment 122 (2001) 1685–1694
Hornsby, P.J., 1995. Biosynthesis of DHEAS by the human adrenal cortex and its age-related decline. Ann. NY Acad. Sci. 774, 29 –46. Hornsby, P.J., 2001. Cell proliferation in mammalian aging. In: Masoro, E.J., Austad, S.N. (Eds.), Handbook of the Biology of Aging, 5th edn. Academic Press, San Diego, CA, pp. 207 – 245. Hornsby, P.J., McAllister, J.M., 1991. Culturing steroidogenic cells. In: Waterman, M.R., Johnson, E.F. (Eds.), Methods in Enzymology, vol. 206. Academic Press, San Diego, CA, pp. 371 – 380. Hornsby, P.J., Aldern, K.A., Harris, S.E., 1986. Clonal variation in response to adrenocorticotropin in cultured bovine adrenocortical cells: relationship to senescence. J. Cell. Physiol. 129, 395 – 402. Hornsby, P.J., Cheng, C.Y., Lala, D.S., Maghsoudlou, S.S., Raju, S.G., Yang, L., 1992. Changes in gene expression during senescence of adrenocortical cells in culture. J. Steroid Biochem. Mol. Biol. 43, 385–395. Kim, N.W., Piatyszek, M.A., Prowse, K.R., et al., 1994. Specific association of human telomerase activity with immortal cells and cancer. Science 266, 2011 – 2015. Martin, G.M., Sprague, C.A., Epstein, C.A., 1970. Replicative life-span of cultivated human cells: effects of donor’s age, tissue and genotype. Lab. Invest. 23, 86 – 92. McAllister, J.M., Hornsby, P.J., 1987. Improved clonal and non-clonal growth of human, rat, and bovine adrenocortical cells in culture. In Vitro Cell. Dev. Biol. 23, 677 – 685. Neville, A.M., O’Hare, M.J., 1982. The Human Adrenal Cortex. Pathology and Biology —an Integrated Approach. Springer, Berlin. Noda, A., Ning, Y., Venable, S.F., Pereira-Smith, O.M., Smith, J.R., 1994. Cloning of senescent cell-derived inhibitors of DNA synthesis using an expression screen. Exp. Cell Res. 211, 90 – 98. Ouellette, M.M., Liao, M., Herbert, B.S., et al., 2000. Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase. J. Biol. Chem. 275, 10072 – 10076. Parker, C.R., Mixon, R.L., Brissie, R.M., Grizzle, W.E., 1997. Aging alters zonation in the adrenal cortex of men. J. Clin. Endocrinol. Metab. 82, 3898 – 3901. Salk, D., 1985. Werner syndrome: a review of recent research with an analysis of connective tissue metabolism, growth control of cultured cells, and chromosomal aberrations. In: Salk, D., Fujiwara, Y., Martin, G.M. (Eds.), Werner’s Syndrome and Human Aging. Plenum, New York, pp. 121 – 160. Schulz, V.P., Zakian, V.A., Ogburn, C.E., et al., 1996. Accelerated loss of telomeric repeats may not explain accelerated replicative decline of Werner syndrome cells. Hum. Genet. 97, 750 – 754. Smith, J.R., Pereira-Smith, O.M., 1996. Replicative senescence: implications for in vivo aging and tumor suppression. Science 273, 63 –67. Suwa, T., Yang, L., Hornsby, P.J., 2001. Telomerase activity in primary cultures of bovine and human adrenocortical cells. J. Endocrinol., in press. Thomas, M., Northrup, S.R., Hornsby, P.J., 1997. Adrenocortical tissue formed by transplantation of normal clones of bovine adrenocortical cells in scid mice replaces the essential functions of the animals’ adrenal glands. Nat. Med. 3, 978 – 983.