REVIEW ABSTRACTS:

J. Campisi. "The Biology of Replicative Senescence." Eur.J.Cancer, Vol. 33, No 5, pp. 703-709, 1997.

Most cells cannot divide indefinitely due to a process termed cellular or replicative senescence. Replicative senescence appears to be a fundamental feature of somatic cells, with the exception of most tumour cells and possibly certain stem cells. How do cells sense the number of divisions they have completed? Although it has not yet been critically tested, the telomere shortening hypothesis is currently perhaps the best explanation for a cell division 'counting' mechanism: Why do cells irreversibly cease proliferation after completing a finite number of divisions? It is now known that replicative senescence alters the expression of a few crucial growth-regulatory genes. It is not known how these changes in growth-regulatory gene expression are related to telomere shortening in higher eukaryotes. However, lower eukaryotes have provided several plausible mechanisms. Finally, what are the physiological consequences of replicative senescence? Several lines of evidence suggest that, at least in human cells, replicative senescence is a powerful tumour suppressive mechanism. There is also indirect evidence that replicative senescence contributes to ageing. Taken together, current findings suggest that, at least in mammals, replicative senescence may have evolved to curtail tumorigenesis, but may also have the unsdected effect of contributing to age-related pathologies, induding cancer.

 Judith Campisi. "Aging and Cancer: The Double-Edged Sword of Replicative Senescence" J Am Geriatr Soc 45:482-488, 1997.

Normal cells do not divide indefinitely. This trait, termed the finite replicative life span of cells, limits the capacity for cell division by a process termed cellular or replicative senescence. Replicative senescence is thought to be a tumor suppression mechanism and also a contributor to organismic aging. This article reviews what is known about the genetics and molecular biology of cell senescence. It discusses the evidence that replicative senescence suppresses tumorigenesis, at least in young organisms, and that it also contributes to the aging of mitotic tissues. Finally, it puts forth the somewhat unorthodox view that, in older organisms, senescent cells may actually contribute to carcinogenesis.

Judith Campisi. The Role of Cellular Senescence in Skin Aging" Journal of Investigative Dermatology Symposium Proceedings 3:1-5, 1998

Higher organisms contain two types of cells: postmitotic cells, which never divide, and mitotic (or mitotically competent) cells, which can divide. Postmitotic cells include mature nerve, muscle, and fat cells, some of which persist for life. Mitotic cells include epithelial and stromal cells of organs such as the skin. Because postmitotic and mitotic cells differ in their proliferative capacity, they may age by different mechanisms. Normal somatic mitotically competent cells do not divide indefinitely. The process that limits the cell division number is termed cellular or replicative senescence. Replicative senescence is thought to be a powerful, albeit imperfect, tumor suppressive mechanism. It is also thought to contribute to organismic aging. Senescent cells undergo three phenotypic changes: they irreversib1y arrest growth, they acquire resistance to apoptotic death, and they acquire altered differentiated functions. The growth arrest is very likely critical for the role of replicative senescence in tumor suppression, but may be less important for the aging of organs such as the skin. On the other hand, the altered differentiation may be critical for compromising the function and integrity of organs like the skin during aging. Senescent keratinocytes and fibroblasts appear to accumulate with age in human skin. Moreover, senescent cells express genes that have long-range, pleiotropic effects - degradative enzymes, growth factors, and inflammatory cytokines. Thus, relatively few senescent cells might compromise skin function and integrity. Moreover, by altering the tissue microenvironment, senescent cells may also contribute to the rise in cancer that occurs with age.

Judith Campisi. "Replicative Senescence and Immortalization" Chapter 8 of The Molecular Basis of Cell Cycle and Growth, G.S. Stein, R. Baserga, A. Giordano, and D.T. Denhardt, ed. NY: Wiley. 1999. pp. 348-373

Normal somatic cells do not divide indefinitely. This property is termed the finite replicative life span of cells, and it restricts cell division by a process known as replicative or cellular senescence. Senescent cells irreversibly arrest proliferation with a G, DNA content. Senescent cells also become resistant to apoptotic death and show selective changes in differentiated cell functions. Several lines of evidence suggest that replicative senescence is a powerful albeit imperfect, tumor suppressive mechanism and that it also contributes to organismic aging. Replicative senescence appears to be controlled by multiple, dominant-acting genetic loci. Cells escape senescence and acquire an infinite replicative life span, or an immortal phenotype, due to a loss of gene function or genetic loci, some of which have been mapped to specific human chromosomes.

What determines replicative life span? What prevents cell division and alters differentiation once replicative capacity is exhausted? Cell division may be limited by progressive telomere shortening. A critically short telomere may induce the expression of dominant growth inhibitors, which institute and maintain the growth arrest. Presenescent and senescent cells express many genes in common. However, some genes whose activities are needed for cell cycle progression are repressed in senescent cells, whereas at least tv~o inhibitors of cell cycle progression are overexpressed by senescent cells. Whether and how these changes in the expression of cell cycle regulatory genes participate in the senescence-associated changes in differentiation is not yet known. Nonetheless, both the changes in cell proliferation and the changes in differentiation that are the hallmark of replicative senescence are likely to have physiological consequences, particularly for mammalian organisms.

Judith Campisi. "Cancer, Aging and Cellular Senescence" In vivo 14:183-188 (2000)

Abstract. Normal cells do not divide indefinitely due to a process termed cellular or replicative senescence. Several lines of evidence suggest that replicative senescence evolved to protect higher eukaryotes, particularly mammals, from developing cancer. Senescent cells differ film their presenescent counterparts in three way: 1) they arrest growth and cannot be stimulated to reenter the cell cycle by physiological mitogens; 2) they become resistant to apoptotic cell death; 3) they acquire altered differentiated functions. Replicative senescence occurs because, owing to the biochemistry of DNA replication, cells acquire one or more critically short telomere. The mechanism by which a short telomere induces the senescent phenotype is unknown. Recent findings suggest that certain types of DNA damage and inappropriate mitogenic signals can also cause cells to adopt a senescent phenotype. Thus, cells respond to a number of potentially oncogenic stimuli by adopting a senescent phenotype. These findings suggest that the senescence response is a failsafe mechanism that protects cells from tumorigenic transformation. Despite the protection from cancer conveyed by cellular senescence and other mechanisms that suppress tumorigenesis, the development of cancer is almost inevitable as mammalian organisms age. Why is this the case? Certainly, aging predisposes cells to accumulate mutations, several of which are necessary before malignant transformation occurs, particularly in humans. However, many benign or relatively well-controlled tumors may also harbor many potentially oncogenic mutations, suggesting that the tissue microenvironment can suppress the expression of many malignant phenotypes. Although the idea remains controversial, cellular senescence has also been proposed to contribute to organismal aging. Senescent cells have recently been shown to accumulate with age in human tissues. One possibility is that the tissue microenvironment is disrupted by the accumulation of dysfunctional senescent cells. Thus, mutation accumulation may synergize with the accumulation of senescent cells, leading to increasing risk for developing cancer that is a hallmark of mammalian aging.

Shurong Huang, Baomin Li, Matthew D. Gray, Junko Oshima , I. Saira Mian & Judith Campisi. " The premature ageing syndrome protein, WRN, is a 3' -> 5' exonuclease" Nature Genetics. Oct. 1998, 20:114-116.

Werner syndrome (WS) is a human autosomal recessive disorder that causes the premature appearance of a partial array of disorders characteristic of old age(1,2). These disorders include atherosclerosis, cancer, type 2 diabetes, osteoporosis, cataracts, wrinkled skin and gray hair, among other ailments. Cells cultured from WS subjects have a shortened replicative life span(3,4) and elevated rates of chromosome translocations, large deletions and homologous recombination(5,6). The gene defective in WS, WRN, encodes a large RecQ-like DNA helicase(7) of 1432 ea. Defects in another human RecQ-like helicase, BLM, result in Bloom's syndrome (8) (BS), a genetic disorder that is quite different from WS. BS is manifested by short stature, neoplasia, immunodeficiency and high risk of cancer. Cells from BS subjects show an increase in sister chromatid exchanges.

DNA helicases can function in replication, repair, recombination, transcription or RNA processing. As WRN and BLM share no obvious homology outside the helicase domain, the non-helicase domains probably determine in which process each RecQ- like helicase participates, which provides a basis for the disparate cellular and organismal phenotypes that result from defects in these proteins.

Statistical sequence analyses showed subtle but significant similarities between WRN and several 3' -> 5' exonucleases(9, 10). To test the prediction that WRN is an exonuclease, we produced tagged recombinant wild-type and mutant WRN proteins. Two mutants had amino-acid substitutions at either position 82 (D82A) or 84 (E84A), two of the five residues predicted to be critical for exonuclease activity (9,l0). A third mutant had a substitution at position 577 (K577M), which abolished WRN helicase activity (11). The fourth mutant was an N-terminal fragment (aa 1-333; N333) containing the putative exonuclease domain, but lacking the helicase domain. A tagged 36-aa vector derived peptide served as a negative control (mock).

Purified WRN and mock proteins were incubated with doubled-stranded DNA substrates. Wild-type WRN degraded a 5' labelled substrate to a series of smaller, labelled products (Fig. 1a), and a 3' labelled substrate to a single labelled product that migrated as a mononucleotide (Fig. 1b). Thus, WRN degraded DNA with 3' -> 5' directionality. Although mock and full-length WRN preparations contained low levels of a contaminating 5 -> 3' exonuclease, as shown by release of the 5' label as a mononucleotide (Figs 1a,2b), 3' -> 5' degradation was entirely dependent on WRN.

WRN exonuclease activity resided in the N terminus. N333, which was essentially free of contaminating 5' -> 3' exonuclease, degraded 5' and 3' labelled substrates similarly to full-length WRN (Fig. 1c,d). When incubated with a 374-bp DNA fragment labelled at the 3' end with 32p, and internally with 3H, N333 released most of both labels (Fig. 1e). Thus, the WRN exonuclease is capable of substantial DNA degradation. Consistent with 3 -> 5' directionality, N333 released 32p from 3' ends more rapidly than 3H from internal residues. In addition, gel-purified N333, which lacked contaminating nuclease activities, efficiently removed the 3', but not the 5', label when incubated with DNA substrates labelled at either the 3' or the 5' end (Fig. 1p.

Genetic evidence for WRN exonuclease activity was obtained by introducing point mutations at critical amino acids in the exonuclease domain (D82A and E84A). These mutants retained the wild-type level of helicase activity (Fig. 2a), but had little or no 3 -> 5' exonuclease activity, using either a 5' (Fig. 2b) or 3' (Fig. 2c) 32p labelled substrate, and were indistinguishable from mock protein in this regard (Fig. 2d). The K577M mutant, in contrast, was devoid of helicase activity (Fig. 2a), as expected, but had 3 -> 5' exonuclease activity comparable to that of wild-type WRN (Fig. 2b-d).

Our data indicate that WRN is indeed a 3' -> 5 exonuclease. This activity resides in the N terminus, and is physically and functionally separable from the helicase activity The identification of an exonuclease activity in WRN clearly distinguishes it from other human RecQ-like helicases, and may help explain the differences between WS and BS.

What are the functions of the WRN exonuclease in vivo? It may participate in recombination and DNA repair. Exonucleases are integral components of some recombination pathways (12), and WRN appears to have a role in recombination (5,6,13). The finding that WS cells are hypersensitive to the DNA damaging agent 4-nitroquinoline-1-oxide (14) suggests a role for WRN in DNA repair. Finally, WRN is homologous to FFA-1 (replication focus-forming activity l) in Xenopus laevis (15), raising the possibility that WRN may also be involved in DNA replication. In this context, the WRN exonuclease may provide 3 -> 5' proofreading function to DNA polymerases that lack such activity. Whatever the case, an understanding of the functions of WRN exonuclease and their relationships to the other functions of WRN will lead to new insights into the molecular and cellular basis for WS and a subset of age-associated pathologies.

1. Martin. G.M. Birth Defects 14, 5-39 (1978).
2. Goto, M. Mech. Ageing Dev 98, 239-254 (1997).
3. Martin, G.M., Sprague, C.A. & Epstein, C.J. Lab.
4. Salk, D., Bryant, E., Hoehn H. Johnston, P. & Martin, G.M. Adv. Exp. ;Bio Med. 190. 305-311 (1985).
5. Fukuchi, K., Martin, G.M. & Monnat R.J. Proc. Natl Acad Sci. USA 86,, 5893-5897 (1989).
6. Cheng, R.Z., Murano, S., Kurz, B. & Shmookler-Reis, R.J. Mutat. Res. 237, 259-269 (1990).
7. Yu, C.E. et.al. Science 272, 258-262 (1996).
8. German. J. Medicine 72, 393-406 (1993).
9. Main, 1.5. Nucleic Acids Res. 25, 3187-3195 (1997).
10. Mushegian, A.R., Bassett, D.E. Jr, Boguski, M.S., Bork, P., & Koonin, E.V Proc. Natl Acad. Sci. USA 94, 5831-5836 (1997).
11. Gray, M.D. et al.. Nature Genet 17, 100-103 (1997).
12. Linn, S.M., Lloyd, R.S. & Roberts, R.T. Nucleases (Cold Spring Harbor Laboratory Press, New York, 1993).
13. Yamagata, K. et al. Proc. Natl Acad Sci. USA 95, 8733-8738 (1998).
14. Ogburn, C.E. et al. Elum. Genet 101, 121-125 (1 997).
15. Yan, H., Chen, C.-Y., Kobayashi, R. & Newport, J. Nature Genet 19, 375-378 (1998).

Judith Campisi. "Replicative Senescence: An Old Lives' Tale?" Cell, Vol. 84, 497-500, February 23, 1996

INTRODUCTION: Normal animal cells, with few exceptions, do not divide indefinitely. This property, termed the finite replicative life span of cells, leads to an eventual arrest of cell division by a process termed cellular or replicative senescence.

Although predicted and observed earlier, replicative senescence was first formally described over 30 years ago when Hayflick and his colleagues reported that human fibroblasts gradually and inevitably lost their ability to proliferate upon continual subculture (Hayflick, 1965). Since then, many cell types from many animal species have been shown to have a finite replicative life span (see Stanulis-Praeger, 1987). Most of these studies have used cells in culture. However, a limited number of in vivo experiments, as well as the evidence discussed here and elsewhere (Stanulis-Praeger, 1987; Campisi et al., 1996), strongly suggest that cellular senescence is not an artifact of culture.

Selected Readings:

-Allsopp, R.C., Vaziri, H., Patterson, C., Goldstein, S., Younglai, E.V., Futcher, A.B., Greider, C.W., and Harley, C.B. (1992). Proc. Natl. Acad. Sci. USA 89, 10114-10118.
-Campisi, J., Dimri, G., and Hara, E. (1996). In Handbook of the Biology of Aging, Fourth Edition, E. Schneider and J. Rowe, eds. (New York: Academic Press), pp. 121-149.
-Carrel, A. (1912). J. Exp. Med. 15, 516-528.
-Choi, H.S., Lin, Z., Li, B.S., and Liu, A.Y. (1990). J. Biol. Chem. 265, 18005-18011.
-Cristofalo, V.J., and Pignolo, R.J. (1993). Physiol. Rev. 73, 617-638.
- Dimri, G.P., Lee, X., Basile, G., Acosta, M., Scott, G., Roskelley, C., Medrano, E., Linskens, M., Rubelj, l., Pereira-Smith, O., Peacocke, M., and Campisi, J. (1995). Proc. Natl. Acad. Sci. USA 92,9363-9367.
-Fawcett, T.W., Sylvester, S.L., Sarge, K.D., Morimoto, R.I., and Holbrook, N.J. (1994). J. Biol. Chem. 269, 32272-32278.
-Goldstein, S. (1990). Science. 249,1129-1133.
-Harley, C.B., and Villeponteau, B. (1995). Curr. Opin. Genet. Dev. 5, 249-255.
-Hayflick, L. (1965). Exp. Cell Res. 37, 614-636.
-Heydari, A.R., Wu, B., Takahashi, R., Strong, R., and Richardson, A. (1993). Mol. Cell. Biol. 13, 2909-2918.
-Jazwinski, S.M. (1993). Genetica 91, 35-51.
- Kenyon, C. (1996). Cell 84, this issue.
-Linskens, M.H., Harley, C.B., West, M.D., Campisi, J., and Hayflick, L. (1995). Science 267, 17.
-Martin, G.M. (1993). J. Gerontol. 48, 171-172.
-Martin, G.M., Sprague, C.A., and Epstein, C.A. (1970). Lab. invest. 23, 86-92.
-McCormick, J.J., and Maher, V.M. (1988). Mutat. Res. 199,273-291.
-Oshima, J., Yu, C.E., Boehnke, M., Weber, J.L., Edelhoff, S., Wagner, M.J., Wells, D.E., Wood, S., Disteche, C.M., Martin, G.M., et al. (1994). Genomics 23, 100-113.
-Oshima, J., Campisi, J., Tannock, T.C., and Martin, G.M. (1995). J. Cell. Physiol. 162, 277-283.
-Rohme, D. (1981). Proc. Natl. Acad. Sci. USA 78, 5009-5013.
-Stanulis-Praeger, B. (1987). Mech. Aging Dev. 38,1-48.
-Wang, E. (1995). Cancer Res. 55, 2284-2292.
-Warner, H.R., Fernandes, G., and Wang, E. (1995). J. Gerontol. 50A, B107-B109.
-Wright, W.E., and Shay, J.W. (1995). Trends Cell Biol. 5, 293-297.

 Sahn-ho Kim, Patrick Kaminker & Judith Campisi. "TIN2, a new regulator of telomere length in human cells" Nature Genetics. Dec.1999, 23: 405-412

Telomeres are DNA-protein structures that cap linear chromosomes and are essential for maintaining genomic stability and cell phenotype. We identified a novel human telomere-associated protein, TIN2, by interaction cloning using the telomeric DNA-binding-protein TRF1 as a bait. TIN2 interacted with TRF1 in vitro and in cells, and colocalized with TRF1 in nuclei and metaphase chromosomes. A mutant TIN2 that lacks amino-terminal sequences effects elongated human telomeres in a telomerase-dependent manner. Our findings suggest that TRF1 is insufficient for control of telomere length in human cells, and that TIN2 is an essential mediator of TRF1 function.

BACK TO TOP