Annals of Medicine
ISSN: 0785-3890 (Print) 1365-2060 (Online) Journal homepage: http://www.tandfonline.com/loi/iann20
Cellular Senescence Katri Koli & Jorma Keski-oja To cite this article: Katri Koli & Jorma Keski-oja (1992) Cellular Senescence, Annals of Medicine, 24:5, 313-318, DOI: 10.3109/07853899209147829 To link to this article: http://dx.doi.org/10.3109/07853899209147829
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Cellular Senescence Katri Koli and Jorma Keski-Oja
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The ageing of cells, cellular senescence, is an event that is encountered in all normal cells. Cells grown in vitro have a limited life span and do not grow well after a certain number of divisions. They cease to divide and eventually die. In accordance with this, the life expectancy of an established cell culture depends on the age of the donor. Cells that have undergone immortalization via a crisls period of transformation by chemicals or viruses, as well as malignant cell lines in general, have an ability to divide indefinitely. A distinct form of cell death, apoptosis or programmed cell death, is encountered in many physiological situations like in keratinocyte differentiation. Key words: cellular senescence; ageing; apoptosis; differentiation; growth regulation. (Annals of Medicine 24: 313-316,1992) In living organisms cell proliferation and cell death are finely balanced. During the last decade our knowledge of mitogenic events and the regulation of cell replication has grown rapidly. Not until now have scientists become aware of the important concept of naturally occurring cell death, which may take place by multiple mechanisms. Programmed cell death, also called apoptosis, is essential for organization of cell associations in developing tissues. In addition, in some adult tissues where cells proliferate continuously, the deletion of old cells is important for regulation of the cell population. In pathological conditions where tissue damage occurs, the removal of injured cells and their replacement with new ones is needed. Necrosis, the classical form of cell death, and accompanying inflammatory response are often observed in tissue insults. Cellular ageing (senescence) is a distinct process leading to cell death (I -3). Limited capacity to proliferate is a widespread phenomenon observed in normal somatic cells of higher eukaryotes. After a defined number of population doublings cells lose their ability to replicate, even in the presence of strong mitogenic stimuli. Senescent cells may stay viable for months and are metabolically active before they eventually die. Senescence has been studied mainly using in vitro cell culture models and the underlaying mechanisms are mostly unknown. The biochemical and genetic events associated with cell senescence involve mechanisms that are encountered in cellular growth control, differentiation and tumourigenesis. From the Departments of Virology (KK, JK-0) and of Dermatology and Venereology (JK-0), University of Helsinki, Finland. Address and reprint requests: Katri Koli, Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki. Finland.
Cellular Senescence Earlier work over 25 years ago using human fibroblast cultures has shown that somatic cells have an inherent limit of proliferative potential. The proliferative capacity was found to be inversely correlated to the age of the tissue donor (4-6). In addition, cell cultures from patients suffering from premature ageing syndromes (progeria) show a decreased life span (7, 8). The proliferation ceases after a certain number of population doublings rather than after a certain chronological age. How this biological clock functions is still a mystery. Much of the earlier information has accumulated from studies with fibroblasts and some biochemical changes observed in senescent cell cultures are also associated with old cells in vivo (3), which is relevant when studying the relationship between in vitro and in vivo ageing. Recently other cell culture models using different cell types have been developed to relate senescence to differentiation pathways, development and tumourigenesis. Senescence is observed in many different species. Senescence at the cellular level can be a manifestation of organismal ageing. This view stems from the fact that the maximum number of doublings of a cell population is generally inversely proportional to the age of the organism and directly proportional to the maximum life span of the species (9). The relevance of in vitro senescence of cell cultures in studying the in vivo ageing process is under some debate. Sometimes immortal cell lines arise spontaneously from normal cells and it has been observed that cells from some species like rodents are more susceptible to escape from senescence by undergoing a crisis period. Clones with an unlimited life span derived from human cells arise very seldom, and this phenomenon can be correlated to resistance to neo-
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plastic transformation (10). Senescence can be considered as a way to suppress tumourigenesis; escape from senescence might be required for tumour growth and metastasis. The relationships between tumour suppressor genes and genes that regulate cellular senescence are interesting from the point of view of the occurrence of cancer during ageing.
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Culture Models for Studies on Cellular Senescence In Vitro Most studies on cellular senescence in viffo have been carried out using fibroblasts, mostly because of the ease of growing them in culture. During senescence not only proliferation ceases, but changes occur also in cell differentiation and other functions, which makes the system more complicated. Specific markers for differentiation of fibroblasts are not available, and the senescent state might be considered as a terminally differentiated fibroblast. Studies with other cell types have given similar results. Techniques for the cultivation of vascular endothelial cells and smooth muscle cells have already been developed. These cells are relatively easy to isolate and many phenotypic markers have been characterized, which makes them a suitable model for studying cell senescence. In addition, hepatocytes and neuronal cells, such as glial cells and neurons, have been used in these studies. Relationships between cell differentiation and senescence have been studied using these model cultures. However, in order to serve as a proper model for senescence the cell culture must retain its identifiable in vivo tissue specific function. Advances in culturing epidermal keratinocytes have made it possible to obtain fully stratified epithelium in vitfo (1 1). Senescent keratinocytes show many characteristics of senescent fibroblasts and undergoing studies are directed to understand the physiological and biochemical changes associated with differentiation of normal and aged keratinocytes. Bovine adrenocortical epithelial cells offer another useful differentiation model, and changes in proliferation and expression of differentiation markers have been characterized in individual cells (12). Another approach to cell senescence is experimentation using somatic cell hybrids. Fusion of immortal cells with normal cells having a limited life span has given some insight to the molecular basis for senescence (13, 14). Characteristics of senescence seem to dominate since such hybrids often have a limited proliferative capacity. Cell hybrids of immortal cells can also produce clones with a limited life span which suggests that immortalization can occur by different mechanisms; otherwise no complementation would occur. The molecular genetics of senescence, studied by microinjecting human chromosomes into immortal cell lines indicated that specific chromosomes contain genes that regulate cell senescence (15). Techniques of molecular biology will certainly open new views also in this field.
Growth Arrest in Senescent Cells Regardless of tissue origin, cells undergoing senescence show some common characteristics. The most striking feature of senescent cells is that they are unable to replicate their DNA and thus enter a new cell cycle. Cells arrest in cycle with a G, DNA content (16). In contrast to quiescent cells, serum or any other mitogenic stimulus fails to initiate DNA synthesis. However, senescent cells are viable and have a functional DNA repair system and active RNA and protein synthesis. Cellular DNA synthesizing machinery of senescent cells under certain circumstances is able to replicate DNA, and the mechanism of the block in the initiation step of DNA synthesis is unknown. The fidelity of DNA polymerase-a, which is the main enzyme responsible for DNA synthesis, decreases as in vitro age increases, and as changes in the structure and genomic organization of DNA occur (17). A significant reduction in ribonucleotide reductase enzyme, which synthesizes the DNA precursors, has been found in association with senescent cells (18). Some DNA tumour viruses are able to overcome the block in initiation of DNA synthesis in senescent cells (19).This ability has been addressed to the transforming proteins of SV40 (large T-antigen), papilloma virus (E6, E7) and adenovirus (ElA, EIB). When senescent cells are transfected with one of these viruses or specifically with the transforming proteins cell proliferation starts, but only a limited increase in life span is achieved. Immortal cell lines can arise after a similar crisis period as seen in normal cells. The transforming mechanisms of T-antigen have been studied extensively and some important clues have been found. Tumour suppressor gene products Rb (retinoblastoma) and p53 proteins have important functions in cell cycle control. T-antigen binds to both Rb and p53, it inhibits their growth-suppressive activity and allows cells to enter the S phase of the cell cycle (20,Zi). The phosphorylation of Rb protein regulates its function during the cell cycle so that the underphosphorylated form has the suppressive activity (22). Senescent cells are not able to phosphorylate Rb protein in response to mitogenic stimuli (23).The Rb protein is among the many substrates for cdc2 kinase, which is essential for the initiation of DNA synthesis and the completion of mitosis. Down-regulation of this kinase during senescence has been observed. The transfection of cdc2 kinase into senescent cells does not overcome the block in DNA synthesis and other cell cycle control mechanisms are probably also involved (24). Recently, it has been shown that cyclins participate actively in the phosphorylation events by interacting with different kinases associated with cell cycle regulation; their down-regulation has also been observed in senescent cells.
Biochemistry of Cellular Senescence Although senescent cells do not divide they are metabolically active. Morphological changes are observed and cells are typically larger with alterations in subcellular architecture (25). Increase in the number of large lyso-
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Cellular Senescence somes and mitochondrial mass are associated with aged cells. The amount of actin increases, and cytoskeletal structures are more rigid with loss of polarity (26). Senescent cells seem also to be more sensitive to contact inhibition. Cell-matrix contacts of old fibroblasts change so that more glycosaminoglycans accumulate to pericellular matrix and more fibronectin with altered structure is synthesized (27, 28). These changes may all contribute to the inability of a cell to divide. Many of the features reported to change with cellular ageing are membrane associated. Gap junctions, which function to change information between neighbouring cells, decrease in number and change morphologically (29). Cell membranes also show increased permeability and alterations in lipid composition, which can be the cause for change in membrane-associated enzymatic activities observed in senescent,cells. The function of growth factor receptors residing in plasma membrane have been studied extensively, since senescent cells cannot respond to mitogenic stimuli. No alterations in the numbers or affinities of the receptors for platelet-derived growth factor, epidermal growth factor or insulin-like growth factor have been observed, and now it is also known that intracellular parts of the signalling pathways are functional (30). In senescent cells protein synthesis is, in general, decreased. The protein content of the cells increases as the cell size increases indicating that there must also be a decline in protein degradation. The major proteins synthesized by senescent cells are similar to those of younger cells. Only a few changes in the expression of specific proteins have been observed. The efficiency of translation of ornithine decarboxylase, a rate-limiting enzyme in polyamine synthesis, is decreased although the expression of mRNA increases (31). Down-regulation of ribosomal structural proteins and changes in the type of histone proteins and mitochondrial proteins expressed have been observed (1). There are no major differences in the changes occurring in senescent and growtharrested cells. Post-transcriptional events like phosphorylation ad glycosylation play also a role in regulating protein function and activity but such changes are not easily detectable. The synthesis of all RNA species decreases with cellular ageing. However, like the protein content, the RNA content of cells increases because of the larger volume of senescent cells. The transcription of some genes to mRNAs changes strikingly during the process of ageing. The proto-oncogene c-fos, whose transcription is transiently induced in cells responding to mitogenic stimuli, is in complete repression in senescent cells (32).This might be an important cause of growth arrest in senescent cells. The expression of some other early genes, like c-myc and c-Ha-ras, is similar in young and senescent cells although they participate in cell proliferation.
Genetics of Cellular Senescence The molecular basis for cellular senescence is still unknown. Currently there are two hypotheses explaining
senescence. One postulates that accumulation of random errors and damage to DNA that cellular proofreading machinery cannot repair, triggers cells to synthesize inhibitors of growth (33). Another hypothesis tries to understand senescence as an active genetic programme, which is dependent on activation or inactivation of specific genes (14,1534). The major open question is whether the inhibitors are the primary cause of senescence or a secondary effect induced by DNA damage or other cellular events. Increasing amounts of evidence indicate that senescence is a dominant feature of somatic cells. Hybrids of immortal cells and cells of finite life span produce clones that are limited in their proliferative capacity (13, 14). In addition, two immortal cell lines fused together often show limited proliferative capacity. Pereira-Smith and Smith (34) have fused many different immortal cell lines and identified four complementation groups for indefinite division. Cell lines in the same group cannot complement each other in fusion experiments, which suggests that immortalization and escape from senescence occurred via the same mechanisms. These studies indicate that four genes or components are important in the pathway to cellular senescence. Introduction of chromosome 4 to immortal cell lines of one of the four complementation groups resulted in the loss of proliferation and reversion of the immortal phenotype (15). Also, human chromosomes 1 and X seem to participate in the regulation of the senescent phenotype. Further studies concentrate to identify genes specially regulating normal growth control and cellular senescence. In addition, an interesting question is how do these genes relate to genes that are important in the regulation of apoptosis and differentiation? Evidence supporting the genetic basis for senescence is convincing, but some aspects favouring the inhibitor hypothesis should be addressed. There is considerable variation in the growth potentials of closely related cells, and this might indicate that senescence is initiated by random events (35). The inhibitor hypothesis is based on exceeding damage to the cells resulting in inhibitor synthesis. In fact, many changes in the chromosome and DNA structure are readily seen in senescent cells. Immortalization could be explained in part by lack of inhibitor synthesis resulting in the transformed phenotype. Results from hybridization studies could also be due to accumulation of diffusible growth inhibitors from the normal counterpart.
Apoptosis Under a variety of physiological conditions apoptosis, or programmed cell death, is a finely regulated way to eliminate cells that are old, unnecessary or harmful (36, 37). It is thought that apoptotic cell death requires the activation of specific ‘death’ genes although little is known about the activating mechanisms. A specific sequence of events that requires energy and active participation from the dying cell leads to the phagocytosis of cell constituents without accompanying inflamma-
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tory reaction. As the cells dying by apoptotic processes do so singly and are rapidly eliminated, it has been difficult to recognize this form of cell death. Apoptosis is the mechanism by which a variety of cell types are deleted during embryonic development (38). Apoptosis occurs frequently in adult tissues that have a high cell turnover rate, like the liver and assorted lymphoid organs. In the clonal selection processes of both T and B lymphocytes self-antigen recognizing clones are eliminated by apoptotic process (39). Cytotoxic T-lymphocytes and natural killer cells induce apoptosis also in their target cells (40). In addition, many other factors that trigger apoptotic reactions have been discovered; removal of growth factors, physiological regulatory hormones, chemotherapeutic agents, proteases and heatkold shock. The most characteristic feature of an apoptotic cell is that it loses water and shrinks, resulting in an increase in buoyant density (41). The extrusion of water is an energy requiring process where intact mitochondria are needed. Also, the plasma membrane of the cells remains intact in the early stages of apoptosis, with a characteristic bubbling appearance resulting from the budding of vesicles from the endoplasmic reticulum and Golgi apparatus (42). The collapse of cytoskeletal structures is associated with cell shrinkage while cells become rounded and detach from their neighbours and the pericellular matrix. Nuclear condensation is accompanied by cleavage of cellular DNA into nucleosome size fragments, which form the ‘chromatic ladder’ of DNA typically seen in agarose gel electrophoresis of apoptotic cells (43). Finally, the cell fragments into apoptotic bodies, small vesicles containing nuclear fragments and other cellular constituents, which are phagocytosed by neighbouring cells or macrophages. The cleavage of DNA into multiples of 180-200 bp fragments is carried out by a specific endonuclease activity. The chemical features of this enzyme have been characterized but it has not been purified to homogenity or cloned yet. In addition, topoisomerase II may participate in the cleavage process (44). The nuclease is present endogenously in the nucleus of cells having high probability of undergoing apoptosis and it can be activated by calcium or calcium ionophores. The endonuclease activity is inhibited by zinc ions: cells of haematopoietic origin undergo rapidly apoptosis if zinc ions are depleted from the growth medium (36).Cells cultured in calcium-free medium are inhibited from undergoing apoptosis. A small increase in intracellular calcium, which has been associated with the early stages of apoptotic cells, could be an important trigger for the process by activating the nuclease. Calcium might also play a role in the transglutaminase activation that is observed in cells undergoing apoptosis (45). As a consequence of transglutaminase activity, protein shells formed by extensive cross-linking of cytoplasmic and membrane proteins are produced.
morphology has been observed. During the maturation of the lens epithelium and erythrocytes there may be a ‘death programme’ operating similar to that found in lymphoid cells. Recently, it has been described that during epidermal keratinocyte differentiationthe granular layer cells initiate DNA fragmentation into typical nucleosome size apoptotic fragments (46). The calcium and magnesium-dependent endonuclease responsible for cleavage is already present in the nucleus of basal keratinocytes, so the cells may be ‘primed’ to die. McCall and Cohen (46) speculate that basal cells produce ‘death proteins’, including the endonuclease and their inhibitors, decrease of which automatically triggers the apoptotic programme. Moreover, the calcium concentration increases in the upper layers of differentiating cells and they are more vulnerable to membrane leakage (47). The increase in intracellular calcium might activate the keratinocyte endonuclease.
Apoptosis in Terminally Differentiating Keratinocytes
Necrosis
In some terminally differentiating cells apoptotic
Tumour Cell Death by Apoptosis In a growing tumour there is a high loss of cells due to cell migration, cell death or in some cases differentiation. In some tumours necrotic reaction due to hypoxia eliminates cells that are distant to blood vessels. However, there is evidence that apoptosis is a more common form of tumour cell death (48). Mild hypoxia, insufficient levels of growth hormones, or presence of infiltrating lymphocytes might activate the tumour cell apoptosis. Cytotoxic T-lymphocytes and natural killer cells both eliminate tumour cells by inducing apoptosis (40). A pore-forming protein, perforin, is produced by cytotoxic T-lymphocytes, and the influx of calcium ions into target cells through the pores might activate the apoptotic process. Also, therapeutic agents and toxins are known to induce tumour cell death via apoptosis. A mitochondria1 inner membrane protein, bcl-2 protooncogene, has been associated with cell survival. Overexpression of bcl-2 blocks the apoptotic death of pro-B-lymphocyte cell line (49). In follicular B-cell lymphoma a t (14; 18) chromosomal translocation juxtaposes the bcl-2 gene with an immunoglobulin locus, and elevated levels of protein lead to prolonged cell survival but no increase in cell cycling. Expression of the bcl-2 gene has also been found in other cell types where its function is associated with cell survival. Understanding the mechanisms of apoptosis is important for the development of better therapy against tumour masses and there has been a recent important finding. Fasantigen belongs to the protein family of tumour necrosis factor receptors, nerve growth factor receptor and CD40 antigen receptor. Monoclonal antibodies against cell surface Fas-antigen induce apoptotic cell death (50). A similar antibody called Apo-1 was found to regress human B cell tumour transplanted into mice (51). Monoclonal antibodies may thus prove useful alternatives in the treatment of various malignancies.
Necrotic reaction is due to severe trauma or some noxious stimuli to cells, which rapidly lose their integrity.
Cellular Senescence This uncontrolled cell death is characterized by high amplitude swelling of mitochondria followed by breakdown of the plasma membrane (52). The rapid rise in intracellular calcium concentration is due to leakage of calcium ions from the extracellular space. Cells ultimately release their cytoplasmic contents to the extracellular fluid, causing damage to neighbouring cells and generating an inflammatory reaction (53). No evidence for specific signalling pathways are involved. The mechanism of necrosis is very different from apoptotic cell death, earlier called srinkage necrosis because of the typical decrease in cell volume. In necrotic cell death DNA is cleaved non-specifically while in apoptosis there is a distinct cleavage pattern.
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Perspective Our understanding of the basic mechanisms of cellular senescence is widening with the development of molecular biology. Significant similarities have been observed in the regulation mechanisms between normal and malignant cell proliferation, cell differentiation and ageing. Numerous biochemical alterations that have been encountered in senescent cells reflect changes that are consequences of more or less specific genetic events. By understanding the specific mechanisms and alterations in gene expression during cell senescence it will be possible to identify critical differences between control of cell proliferation and growth inhibition, cancerous growth and cell ageing. We thank Dr Marikki Laiho for critical comments.
References 1. Peacocke M, Camplsi J. Cellular senescence: a reflection of normal growth control, differentiation, or aging? J Cell Biochem 1991; 45: 147-55.
2. Stanulis-Praeger BM. Cellular senescence revisited: a review. Mech Ageing Dev 1987; 38: 1-48. 3. Smith JR, Lincoln DW. Ageing of cells in culture. lnt Rev Cytoll984; 89: 151-77. 4. Hayfllck L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 1965; 37: 614-36. 5. Martin GM, Sprague CA, Epstein CJ. Replicative life span of cultivated human cells. Effect of donor's age, tissue and genotype. Lab Invest 1970; 23: 86-92. 6. Schnelder EL, Mitsui Y. The relationship between in vitro cellular ageing and in vivo human age. Proc Natl Acad Sci USA 1976; 73: 3584-8. 7. Goldstein S. Life span of cultured cells in progeria Lancet 1969; 1: 424. 8. Norwood TH, Hoehm H, Salk D, Martin GM. Cellular ageing in Werner's syndrome: a unique phenotype. J Invest Dermatol1979; 72: 92-6. 9. McCormick A, Campisi, J. Cellular ageing and senescence. Curr Opin Cell Biol 1991; 3: 230-4. 10. Milo GE, Casto BC. Conditions for transformation of human fibroblast cells: an overview. Cancer Lett 1986; 31: 1-1 3. 11. Fuchs E. Epidermal differentiation. Curr Opinion Cell Biol 1990; 2: 1028-35.
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12. Hornsby PJ, Hancock JP, V o TP, Nason LM, Ryan RF, McAlllster JM. Loss of expression of a differentiated function gene, steroid 17a-hydroxylase, as adrenocortical cells senesce in culture. Proc NatlAcad Sci USA 1987; 1580-4. 13. Bunn CL, Tarrant GM. Limited life span in somatic cell hybrids and cybribs. Exp Cell Res 1980; 127: 385-96. 14. Perelra-Smith OM, Smith JR. Expression of SV40 T antigen in finite life span hybrids of normal SV40 transformed fibroblasts. Somatic Cell Genet 1981; 7: 41 1-21, 15. Ning Y, Weber JL, Klllary AM, Ledbetter DH, Smith JR, Perelra-Smith OH. Genetic analysis of indefinite division in human cells: evidence for a cell senescence-related gene@)on human chromosome 4. Proc NaN Acad Sci USA 1991; 88:5635-9. 16. Sherwood SW, Rush D, Ellsworth JL, Schimke R. Defining cellular senescence in IMR-90 cells: a flow cytometric analysis. Proc Natl Acad Sci USA 1988; 85:9086-90. 17. Murray V. Properties of DNA polymerases from young and ageing human fibroblasts. Mech Ageing Dev 1981; 16: 327-43. 18. Dick JF, Wright JA. Involvement of ribonucleotide reductase activity in senescence of normal human diploid fibroblasts. Mech Ageing Dev 1982; 20: 103-9. 19. Wright WE, Perelra-Smith OM, Shay JW. Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts. Mol Cell Biol 1989; 9: 3088-92. 20. DeCaprio JA, Ludiow JW, Figge J et al. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 1988; 54: 275-83. 21. Werness SC, Levlne AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990; 248: 76-9. 22. DeCaprio JA, Ludlow JW, Lynch D et al. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 1989; 58: 1085-95. 23. Stein GH, Beeson M, Gordon L. Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts. Science 1990; 249: 666-9. 24. Richter KH, Afsharl CA, Annab LA et al. Down-regulation of cdc2 in senescent human and hamster cells. Cancer Res 1991; 51: 6010-1 3. 25. Simons JWIM. The use of frequency distribution of cell diameters to characterize cell populations in tissue culture. Exp CeN Res 1967; 45: 336-50. 26. Wang E, Gunderson D. Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro. Exp Cell Res 1984; 154: 191-202. 27. Kumazaki T, Robetorye Rs, Robetorye SC, Smith JR. Fibronectin expression increases during in vitro cellular senescence: correlation with increased cell area. Exp Cell Res 1991; 195: 13-1 9. 28. Magnuson VL, Young M, Schattenberg DG et 81. The alternative splicing of fibronectin pre-mRNA is altered during ageing in response to growth factors. J Biol Chem 1991; 266: 14654-62. 29. Kelley RO, Vogel KG, Crissman HA, Lujan CJ, Skipper BE. Development of the ageing cell surface: reduction of gap junction-mediated metabolic cooperation with progressive subcultivation of human embryo fibroblasts (IMR-90). f x p Cell Res 1979; 119: 127-1 43. 30. Paulsson Y, Bywater M, Pfelfer-Ohlsson S et al. Growth factors induce early pre-replicative changes in senescent human fibroblasts. EM80 J 1986; 5: 2157-62. 31. Chang ZF, Chen KY. Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts. J Biol Chem 1988; 263: 11431-5. 32. Seshadri T, Camplsi J. Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science 1990; 247: 205-9. 33. Rosenberger RF, Gounaris E, Kolettas E. Mechanisms responsible for the limited life span and immortal pheno-
318
34.
35. 36.
37. 38. 39.
Downloaded by [Universität Osnabrueck] at 08:26 16 March 2016
40.
41. 42. 43.
Koli
Keski-Oja
types in cultured mammalian cells. J Theor Biol 1991 ; 148: 383-92. Perelra-Smith OM,Smith JR. Genetic analysis of indefinite division in human cells: identification of four complementation groups. Proc Natl Acad Sci USA 1988; 85: 6042-6. McCarron M, Osborne Y, Story CJ, Dempsey JL, Turner DR, Morley AA. Effect of age on lymphocyte proliferation. Mech Ageing Dev 1987; 41: 21 1-1 8. Cotter TG, Lennon SV, Glynn JG, Martin SJ. Cell death via apoptosis and its relationship to growth, development and differentiation of both tumor and normal cells. Anticancer Res 1990; 10: 1 1 53-60. Arends MJ, Wyllle AH. Apoptosis: mechanisms and roles in pathology. Int Rev Exp Patholl991; 32: 23-54. Kerr JFR, Searle J, Harmon BV, Blshop CJ. Apoptosis. In: Potten CS, ed. Perspectives in mammalian cell death. Oxford: Oxford University Press, 1987: 93-128. McDonald HR, Lees RK. Programmed death of autoreactive thymocytes. Nature 1990;343 624-44. Hameed A, Oisen KJ, Lee MK, Llchtenheld MG, Podack ER. Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis. J Exp Med 1989; 169: 765-77. Lockshln RA, Beaulation J. Cell death: questions for histochemists concerning the causes of the various cytological changes. HisfochemJ 1981; 13: 659-66. Stacey NH, Blshop CJ, Halllday JW et al. Apoptosis as a mode of cell death in antibody-dependent lymphocytotoxicity. J Cell Sci 1985; 74: 169-79. Duke RC, Chewenak R, Cohen JJ. Endogenous endonuclease-inducedDNA fragmentation: an early event in cell mediated cytolysis. Proc Natl Acad Sci USA 1983 80: 6361 -5.
44. Chow K-C, Ross WE. Topoisomerase-specific drug sensltivity in relation to cell cycle progression. Mol Cell Biol 1987; 7: 31 19-23. 45..Fesus L, Thomazy V, Falus A. Induction and activation of tissue transglutaminase during programmed cell death. FEBS Lett 1987; 224: 104-8. 46. McCall CA, Cohen JJ. Programmed cell death in terminally differentiating keratinocytes: role of endogenous endonuclease. J lnvest Dermatoll991; 97: 1 1 1-1 4. 47. Whitfield JF. Calcium signals and cancer. Cfit Rev Oncogenesis 1992; 3: 55-90. 48. Szende B, Zalatna A, Schaily AV. Programmed cell death (apoptosis) in pancreatic cancer of hamsters after treatment with analogs of both luteinizing hormone releasing hormone and somatostatin. Proc Natl Acad Sci USA 1989; 8 6 1643-7. 49. Hockenbery D, Nirnez G, Mllllman C, Schrelber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondria1 membrane protein that blocks programmed cell death. Nature 1990; 343: 334-6. 50. ltoh N, Yonehara S, lshll A et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991 ; 66: 233-4. 51. Trauth BC, Klas C, Peters AMJ. Monoclonal antibodymediated tumor regression by induction of apoptosis. Science 1989; 245: 301 -5. 52. Trump BF, Berezesky IK, Osornlo-Vargas AR. Cell death and the disease process. The role of calcium. In: Bowen ID, Lockshin RA, eds. Cell death in biology and pathology. London: Chapman and Hall, 1981 : 209-42. 53. Wyllle AH. Cell death: a new classification separating apoptosis from necrosis. In: Bowen ID, Lockshin RA, eds. Cell death in biology and pathology, London: Chapman and Hall, 1981: 9-34.
ISSN: 0785-3890 (Print) 1365-2060 (Online) Journal homepage: http://www.tandfonline.com/loi/iann20
Cellular Senescence Katri Koli & Jorma Keski-oja To cite this article: Katri Koli & Jorma Keski-oja (1992) Cellular Senescence, Annals of Medicine, 24:5, 313-318, DOI: 10.3109/07853899209147829 To link to this article: http://dx.doi.org/10.3109/07853899209147829
Published online: 08 Jul 2009.
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Review Article
Cellular Senescence Katri Koli and Jorma Keski-Oja
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The ageing of cells, cellular senescence, is an event that is encountered in all normal cells. Cells grown in vitro have a limited life span and do not grow well after a certain number of divisions. They cease to divide and eventually die. In accordance with this, the life expectancy of an established cell culture depends on the age of the donor. Cells that have undergone immortalization via a crisls period of transformation by chemicals or viruses, as well as malignant cell lines in general, have an ability to divide indefinitely. A distinct form of cell death, apoptosis or programmed cell death, is encountered in many physiological situations like in keratinocyte differentiation. Key words: cellular senescence; ageing; apoptosis; differentiation; growth regulation. (Annals of Medicine 24: 313-316,1992) In living organisms cell proliferation and cell death are finely balanced. During the last decade our knowledge of mitogenic events and the regulation of cell replication has grown rapidly. Not until now have scientists become aware of the important concept of naturally occurring cell death, which may take place by multiple mechanisms. Programmed cell death, also called apoptosis, is essential for organization of cell associations in developing tissues. In addition, in some adult tissues where cells proliferate continuously, the deletion of old cells is important for regulation of the cell population. In pathological conditions where tissue damage occurs, the removal of injured cells and their replacement with new ones is needed. Necrosis, the classical form of cell death, and accompanying inflammatory response are often observed in tissue insults. Cellular ageing (senescence) is a distinct process leading to cell death (I -3). Limited capacity to proliferate is a widespread phenomenon observed in normal somatic cells of higher eukaryotes. After a defined number of population doublings cells lose their ability to replicate, even in the presence of strong mitogenic stimuli. Senescent cells may stay viable for months and are metabolically active before they eventually die. Senescence has been studied mainly using in vitro cell culture models and the underlaying mechanisms are mostly unknown. The biochemical and genetic events associated with cell senescence involve mechanisms that are encountered in cellular growth control, differentiation and tumourigenesis. From the Departments of Virology (KK, JK-0) and of Dermatology and Venereology (JK-0), University of Helsinki, Finland. Address and reprint requests: Katri Koli, Department of Virology, University of Helsinki, Haartmaninkatu 3, SF-00290 Helsinki. Finland.
Cellular Senescence Earlier work over 25 years ago using human fibroblast cultures has shown that somatic cells have an inherent limit of proliferative potential. The proliferative capacity was found to be inversely correlated to the age of the tissue donor (4-6). In addition, cell cultures from patients suffering from premature ageing syndromes (progeria) show a decreased life span (7, 8). The proliferation ceases after a certain number of population doublings rather than after a certain chronological age. How this biological clock functions is still a mystery. Much of the earlier information has accumulated from studies with fibroblasts and some biochemical changes observed in senescent cell cultures are also associated with old cells in vivo (3), which is relevant when studying the relationship between in vitro and in vivo ageing. Recently other cell culture models using different cell types have been developed to relate senescence to differentiation pathways, development and tumourigenesis. Senescence is observed in many different species. Senescence at the cellular level can be a manifestation of organismal ageing. This view stems from the fact that the maximum number of doublings of a cell population is generally inversely proportional to the age of the organism and directly proportional to the maximum life span of the species (9). The relevance of in vitro senescence of cell cultures in studying the in vivo ageing process is under some debate. Sometimes immortal cell lines arise spontaneously from normal cells and it has been observed that cells from some species like rodents are more susceptible to escape from senescence by undergoing a crisis period. Clones with an unlimited life span derived from human cells arise very seldom, and this phenomenon can be correlated to resistance to neo-
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plastic transformation (10). Senescence can be considered as a way to suppress tumourigenesis; escape from senescence might be required for tumour growth and metastasis. The relationships between tumour suppressor genes and genes that regulate cellular senescence are interesting from the point of view of the occurrence of cancer during ageing.
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Culture Models for Studies on Cellular Senescence In Vitro Most studies on cellular senescence in viffo have been carried out using fibroblasts, mostly because of the ease of growing them in culture. During senescence not only proliferation ceases, but changes occur also in cell differentiation and other functions, which makes the system more complicated. Specific markers for differentiation of fibroblasts are not available, and the senescent state might be considered as a terminally differentiated fibroblast. Studies with other cell types have given similar results. Techniques for the cultivation of vascular endothelial cells and smooth muscle cells have already been developed. These cells are relatively easy to isolate and many phenotypic markers have been characterized, which makes them a suitable model for studying cell senescence. In addition, hepatocytes and neuronal cells, such as glial cells and neurons, have been used in these studies. Relationships between cell differentiation and senescence have been studied using these model cultures. However, in order to serve as a proper model for senescence the cell culture must retain its identifiable in vivo tissue specific function. Advances in culturing epidermal keratinocytes have made it possible to obtain fully stratified epithelium in vitfo (1 1). Senescent keratinocytes show many characteristics of senescent fibroblasts and undergoing studies are directed to understand the physiological and biochemical changes associated with differentiation of normal and aged keratinocytes. Bovine adrenocortical epithelial cells offer another useful differentiation model, and changes in proliferation and expression of differentiation markers have been characterized in individual cells (12). Another approach to cell senescence is experimentation using somatic cell hybrids. Fusion of immortal cells with normal cells having a limited life span has given some insight to the molecular basis for senescence (13, 14). Characteristics of senescence seem to dominate since such hybrids often have a limited proliferative capacity. Cell hybrids of immortal cells can also produce clones with a limited life span which suggests that immortalization can occur by different mechanisms; otherwise no complementation would occur. The molecular genetics of senescence, studied by microinjecting human chromosomes into immortal cell lines indicated that specific chromosomes contain genes that regulate cell senescence (15). Techniques of molecular biology will certainly open new views also in this field.
Growth Arrest in Senescent Cells Regardless of tissue origin, cells undergoing senescence show some common characteristics. The most striking feature of senescent cells is that they are unable to replicate their DNA and thus enter a new cell cycle. Cells arrest in cycle with a G, DNA content (16). In contrast to quiescent cells, serum or any other mitogenic stimulus fails to initiate DNA synthesis. However, senescent cells are viable and have a functional DNA repair system and active RNA and protein synthesis. Cellular DNA synthesizing machinery of senescent cells under certain circumstances is able to replicate DNA, and the mechanism of the block in the initiation step of DNA synthesis is unknown. The fidelity of DNA polymerase-a, which is the main enzyme responsible for DNA synthesis, decreases as in vitro age increases, and as changes in the structure and genomic organization of DNA occur (17). A significant reduction in ribonucleotide reductase enzyme, which synthesizes the DNA precursors, has been found in association with senescent cells (18). Some DNA tumour viruses are able to overcome the block in initiation of DNA synthesis in senescent cells (19).This ability has been addressed to the transforming proteins of SV40 (large T-antigen), papilloma virus (E6, E7) and adenovirus (ElA, EIB). When senescent cells are transfected with one of these viruses or specifically with the transforming proteins cell proliferation starts, but only a limited increase in life span is achieved. Immortal cell lines can arise after a similar crisis period as seen in normal cells. The transforming mechanisms of T-antigen have been studied extensively and some important clues have been found. Tumour suppressor gene products Rb (retinoblastoma) and p53 proteins have important functions in cell cycle control. T-antigen binds to both Rb and p53, it inhibits their growth-suppressive activity and allows cells to enter the S phase of the cell cycle (20,Zi). The phosphorylation of Rb protein regulates its function during the cell cycle so that the underphosphorylated form has the suppressive activity (22). Senescent cells are not able to phosphorylate Rb protein in response to mitogenic stimuli (23).The Rb protein is among the many substrates for cdc2 kinase, which is essential for the initiation of DNA synthesis and the completion of mitosis. Down-regulation of this kinase during senescence has been observed. The transfection of cdc2 kinase into senescent cells does not overcome the block in DNA synthesis and other cell cycle control mechanisms are probably also involved (24). Recently, it has been shown that cyclins participate actively in the phosphorylation events by interacting with different kinases associated with cell cycle regulation; their down-regulation has also been observed in senescent cells.
Biochemistry of Cellular Senescence Although senescent cells do not divide they are metabolically active. Morphological changes are observed and cells are typically larger with alterations in subcellular architecture (25). Increase in the number of large lyso-
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Cellular Senescence somes and mitochondrial mass are associated with aged cells. The amount of actin increases, and cytoskeletal structures are more rigid with loss of polarity (26). Senescent cells seem also to be more sensitive to contact inhibition. Cell-matrix contacts of old fibroblasts change so that more glycosaminoglycans accumulate to pericellular matrix and more fibronectin with altered structure is synthesized (27, 28). These changes may all contribute to the inability of a cell to divide. Many of the features reported to change with cellular ageing are membrane associated. Gap junctions, which function to change information between neighbouring cells, decrease in number and change morphologically (29). Cell membranes also show increased permeability and alterations in lipid composition, which can be the cause for change in membrane-associated enzymatic activities observed in senescent,cells. The function of growth factor receptors residing in plasma membrane have been studied extensively, since senescent cells cannot respond to mitogenic stimuli. No alterations in the numbers or affinities of the receptors for platelet-derived growth factor, epidermal growth factor or insulin-like growth factor have been observed, and now it is also known that intracellular parts of the signalling pathways are functional (30). In senescent cells protein synthesis is, in general, decreased. The protein content of the cells increases as the cell size increases indicating that there must also be a decline in protein degradation. The major proteins synthesized by senescent cells are similar to those of younger cells. Only a few changes in the expression of specific proteins have been observed. The efficiency of translation of ornithine decarboxylase, a rate-limiting enzyme in polyamine synthesis, is decreased although the expression of mRNA increases (31). Down-regulation of ribosomal structural proteins and changes in the type of histone proteins and mitochondrial proteins expressed have been observed (1). There are no major differences in the changes occurring in senescent and growtharrested cells. Post-transcriptional events like phosphorylation ad glycosylation play also a role in regulating protein function and activity but such changes are not easily detectable. The synthesis of all RNA species decreases with cellular ageing. However, like the protein content, the RNA content of cells increases because of the larger volume of senescent cells. The transcription of some genes to mRNAs changes strikingly during the process of ageing. The proto-oncogene c-fos, whose transcription is transiently induced in cells responding to mitogenic stimuli, is in complete repression in senescent cells (32).This might be an important cause of growth arrest in senescent cells. The expression of some other early genes, like c-myc and c-Ha-ras, is similar in young and senescent cells although they participate in cell proliferation.
Genetics of Cellular Senescence The molecular basis for cellular senescence is still unknown. Currently there are two hypotheses explaining
senescence. One postulates that accumulation of random errors and damage to DNA that cellular proofreading machinery cannot repair, triggers cells to synthesize inhibitors of growth (33). Another hypothesis tries to understand senescence as an active genetic programme, which is dependent on activation or inactivation of specific genes (14,1534). The major open question is whether the inhibitors are the primary cause of senescence or a secondary effect induced by DNA damage or other cellular events. Increasing amounts of evidence indicate that senescence is a dominant feature of somatic cells. Hybrids of immortal cells and cells of finite life span produce clones that are limited in their proliferative capacity (13, 14). In addition, two immortal cell lines fused together often show limited proliferative capacity. Pereira-Smith and Smith (34) have fused many different immortal cell lines and identified four complementation groups for indefinite division. Cell lines in the same group cannot complement each other in fusion experiments, which suggests that immortalization and escape from senescence occurred via the same mechanisms. These studies indicate that four genes or components are important in the pathway to cellular senescence. Introduction of chromosome 4 to immortal cell lines of one of the four complementation groups resulted in the loss of proliferation and reversion of the immortal phenotype (15). Also, human chromosomes 1 and X seem to participate in the regulation of the senescent phenotype. Further studies concentrate to identify genes specially regulating normal growth control and cellular senescence. In addition, an interesting question is how do these genes relate to genes that are important in the regulation of apoptosis and differentiation? Evidence supporting the genetic basis for senescence is convincing, but some aspects favouring the inhibitor hypothesis should be addressed. There is considerable variation in the growth potentials of closely related cells, and this might indicate that senescence is initiated by random events (35). The inhibitor hypothesis is based on exceeding damage to the cells resulting in inhibitor synthesis. In fact, many changes in the chromosome and DNA structure are readily seen in senescent cells. Immortalization could be explained in part by lack of inhibitor synthesis resulting in the transformed phenotype. Results from hybridization studies could also be due to accumulation of diffusible growth inhibitors from the normal counterpart.
Apoptosis Under a variety of physiological conditions apoptosis, or programmed cell death, is a finely regulated way to eliminate cells that are old, unnecessary or harmful (36, 37). It is thought that apoptotic cell death requires the activation of specific ‘death’ genes although little is known about the activating mechanisms. A specific sequence of events that requires energy and active participation from the dying cell leads to the phagocytosis of cell constituents without accompanying inflamma-
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tory reaction. As the cells dying by apoptotic processes do so singly and are rapidly eliminated, it has been difficult to recognize this form of cell death. Apoptosis is the mechanism by which a variety of cell types are deleted during embryonic development (38). Apoptosis occurs frequently in adult tissues that have a high cell turnover rate, like the liver and assorted lymphoid organs. In the clonal selection processes of both T and B lymphocytes self-antigen recognizing clones are eliminated by apoptotic process (39). Cytotoxic T-lymphocytes and natural killer cells induce apoptosis also in their target cells (40). In addition, many other factors that trigger apoptotic reactions have been discovered; removal of growth factors, physiological regulatory hormones, chemotherapeutic agents, proteases and heatkold shock. The most characteristic feature of an apoptotic cell is that it loses water and shrinks, resulting in an increase in buoyant density (41). The extrusion of water is an energy requiring process where intact mitochondria are needed. Also, the plasma membrane of the cells remains intact in the early stages of apoptosis, with a characteristic bubbling appearance resulting from the budding of vesicles from the endoplasmic reticulum and Golgi apparatus (42). The collapse of cytoskeletal structures is associated with cell shrinkage while cells become rounded and detach from their neighbours and the pericellular matrix. Nuclear condensation is accompanied by cleavage of cellular DNA into nucleosome size fragments, which form the ‘chromatic ladder’ of DNA typically seen in agarose gel electrophoresis of apoptotic cells (43). Finally, the cell fragments into apoptotic bodies, small vesicles containing nuclear fragments and other cellular constituents, which are phagocytosed by neighbouring cells or macrophages. The cleavage of DNA into multiples of 180-200 bp fragments is carried out by a specific endonuclease activity. The chemical features of this enzyme have been characterized but it has not been purified to homogenity or cloned yet. In addition, topoisomerase II may participate in the cleavage process (44). The nuclease is present endogenously in the nucleus of cells having high probability of undergoing apoptosis and it can be activated by calcium or calcium ionophores. The endonuclease activity is inhibited by zinc ions: cells of haematopoietic origin undergo rapidly apoptosis if zinc ions are depleted from the growth medium (36).Cells cultured in calcium-free medium are inhibited from undergoing apoptosis. A small increase in intracellular calcium, which has been associated with the early stages of apoptotic cells, could be an important trigger for the process by activating the nuclease. Calcium might also play a role in the transglutaminase activation that is observed in cells undergoing apoptosis (45). As a consequence of transglutaminase activity, protein shells formed by extensive cross-linking of cytoplasmic and membrane proteins are produced.
morphology has been observed. During the maturation of the lens epithelium and erythrocytes there may be a ‘death programme’ operating similar to that found in lymphoid cells. Recently, it has been described that during epidermal keratinocyte differentiationthe granular layer cells initiate DNA fragmentation into typical nucleosome size apoptotic fragments (46). The calcium and magnesium-dependent endonuclease responsible for cleavage is already present in the nucleus of basal keratinocytes, so the cells may be ‘primed’ to die. McCall and Cohen (46) speculate that basal cells produce ‘death proteins’, including the endonuclease and their inhibitors, decrease of which automatically triggers the apoptotic programme. Moreover, the calcium concentration increases in the upper layers of differentiating cells and they are more vulnerable to membrane leakage (47). The increase in intracellular calcium might activate the keratinocyte endonuclease.
Apoptosis in Terminally Differentiating Keratinocytes
Necrosis
In some terminally differentiating cells apoptotic
Tumour Cell Death by Apoptosis In a growing tumour there is a high loss of cells due to cell migration, cell death or in some cases differentiation. In some tumours necrotic reaction due to hypoxia eliminates cells that are distant to blood vessels. However, there is evidence that apoptosis is a more common form of tumour cell death (48). Mild hypoxia, insufficient levels of growth hormones, or presence of infiltrating lymphocytes might activate the tumour cell apoptosis. Cytotoxic T-lymphocytes and natural killer cells both eliminate tumour cells by inducing apoptosis (40). A pore-forming protein, perforin, is produced by cytotoxic T-lymphocytes, and the influx of calcium ions into target cells through the pores might activate the apoptotic process. Also, therapeutic agents and toxins are known to induce tumour cell death via apoptosis. A mitochondria1 inner membrane protein, bcl-2 protooncogene, has been associated with cell survival. Overexpression of bcl-2 blocks the apoptotic death of pro-B-lymphocyte cell line (49). In follicular B-cell lymphoma a t (14; 18) chromosomal translocation juxtaposes the bcl-2 gene with an immunoglobulin locus, and elevated levels of protein lead to prolonged cell survival but no increase in cell cycling. Expression of the bcl-2 gene has also been found in other cell types where its function is associated with cell survival. Understanding the mechanisms of apoptosis is important for the development of better therapy against tumour masses and there has been a recent important finding. Fasantigen belongs to the protein family of tumour necrosis factor receptors, nerve growth factor receptor and CD40 antigen receptor. Monoclonal antibodies against cell surface Fas-antigen induce apoptotic cell death (50). A similar antibody called Apo-1 was found to regress human B cell tumour transplanted into mice (51). Monoclonal antibodies may thus prove useful alternatives in the treatment of various malignancies.
Necrotic reaction is due to severe trauma or some noxious stimuli to cells, which rapidly lose their integrity.
Cellular Senescence This uncontrolled cell death is characterized by high amplitude swelling of mitochondria followed by breakdown of the plasma membrane (52). The rapid rise in intracellular calcium concentration is due to leakage of calcium ions from the extracellular space. Cells ultimately release their cytoplasmic contents to the extracellular fluid, causing damage to neighbouring cells and generating an inflammatory reaction (53). No evidence for specific signalling pathways are involved. The mechanism of necrosis is very different from apoptotic cell death, earlier called srinkage necrosis because of the typical decrease in cell volume. In necrotic cell death DNA is cleaved non-specifically while in apoptosis there is a distinct cleavage pattern.
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Perspective Our understanding of the basic mechanisms of cellular senescence is widening with the development of molecular biology. Significant similarities have been observed in the regulation mechanisms between normal and malignant cell proliferation, cell differentiation and ageing. Numerous biochemical alterations that have been encountered in senescent cells reflect changes that are consequences of more or less specific genetic events. By understanding the specific mechanisms and alterations in gene expression during cell senescence it will be possible to identify critical differences between control of cell proliferation and growth inhibition, cancerous growth and cell ageing. We thank Dr Marikki Laiho for critical comments.
References 1. Peacocke M, Camplsi J. Cellular senescence: a reflection of normal growth control, differentiation, or aging? J Cell Biochem 1991; 45: 147-55.
2. Stanulis-Praeger BM. Cellular senescence revisited: a review. Mech Ageing Dev 1987; 38: 1-48. 3. Smith JR, Lincoln DW. Ageing of cells in culture. lnt Rev Cytoll984; 89: 151-77. 4. Hayfllck L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res 1965; 37: 614-36. 5. Martin GM, Sprague CA, Epstein CJ. Replicative life span of cultivated human cells. Effect of donor's age, tissue and genotype. Lab Invest 1970; 23: 86-92. 6. Schnelder EL, Mitsui Y. The relationship between in vitro cellular ageing and in vivo human age. Proc Natl Acad Sci USA 1976; 73: 3584-8. 7. Goldstein S. Life span of cultured cells in progeria Lancet 1969; 1: 424. 8. Norwood TH, Hoehm H, Salk D, Martin GM. Cellular ageing in Werner's syndrome: a unique phenotype. J Invest Dermatol1979; 72: 92-6. 9. McCormick A, Campisi, J. Cellular ageing and senescence. Curr Opin Cell Biol 1991; 3: 230-4. 10. Milo GE, Casto BC. Conditions for transformation of human fibroblast cells: an overview. Cancer Lett 1986; 31: 1-1 3. 11. Fuchs E. Epidermal differentiation. Curr Opinion Cell Biol 1990; 2: 1028-35.
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12. Hornsby PJ, Hancock JP, V o TP, Nason LM, Ryan RF, McAlllster JM. Loss of expression of a differentiated function gene, steroid 17a-hydroxylase, as adrenocortical cells senesce in culture. Proc NatlAcad Sci USA 1987; 1580-4. 13. Bunn CL, Tarrant GM. Limited life span in somatic cell hybrids and cybribs. Exp Cell Res 1980; 127: 385-96. 14. Perelra-Smith OM, Smith JR. Expression of SV40 T antigen in finite life span hybrids of normal SV40 transformed fibroblasts. Somatic Cell Genet 1981; 7: 41 1-21, 15. Ning Y, Weber JL, Klllary AM, Ledbetter DH, Smith JR, Perelra-Smith OH. Genetic analysis of indefinite division in human cells: evidence for a cell senescence-related gene@)on human chromosome 4. Proc NaN Acad Sci USA 1991; 88:5635-9. 16. Sherwood SW, Rush D, Ellsworth JL, Schimke R. Defining cellular senescence in IMR-90 cells: a flow cytometric analysis. Proc Natl Acad Sci USA 1988; 85:9086-90. 17. Murray V. Properties of DNA polymerases from young and ageing human fibroblasts. Mech Ageing Dev 1981; 16: 327-43. 18. Dick JF, Wright JA. Involvement of ribonucleotide reductase activity in senescence of normal human diploid fibroblasts. Mech Ageing Dev 1982; 20: 103-9. 19. Wright WE, Perelra-Smith OM, Shay JW. Reversible cellular senescence: implications for immortalization of normal human diploid fibroblasts. Mol Cell Biol 1989; 9: 3088-92. 20. DeCaprio JA, Ludiow JW, Figge J et al. SV40 large tumor antigen forms a specific complex with the product of the retinoblastoma susceptibility gene. Cell 1988; 54: 275-83. 21. Werness SC, Levlne AJ, Howley PM. Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 1990; 248: 76-9. 22. DeCaprio JA, Ludlow JW, Lynch D et al. The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element. Cell 1989; 58: 1085-95. 23. Stein GH, Beeson M, Gordon L. Failure to phosphorylate the retinoblastoma gene product in senescent human fibroblasts. Science 1990; 249: 666-9. 24. Richter KH, Afsharl CA, Annab LA et al. Down-regulation of cdc2 in senescent human and hamster cells. Cancer Res 1991; 51: 6010-1 3. 25. Simons JWIM. The use of frequency distribution of cell diameters to characterize cell populations in tissue culture. Exp CeN Res 1967; 45: 336-50. 26. Wang E, Gunderson D. Increased organization of cytoskeleton accompanying the aging of human fibroblasts in vitro. Exp Cell Res 1984; 154: 191-202. 27. Kumazaki T, Robetorye Rs, Robetorye SC, Smith JR. Fibronectin expression increases during in vitro cellular senescence: correlation with increased cell area. Exp Cell Res 1991; 195: 13-1 9. 28. Magnuson VL, Young M, Schattenberg DG et 81. The alternative splicing of fibronectin pre-mRNA is altered during ageing in response to growth factors. J Biol Chem 1991; 266: 14654-62. 29. Kelley RO, Vogel KG, Crissman HA, Lujan CJ, Skipper BE. Development of the ageing cell surface: reduction of gap junction-mediated metabolic cooperation with progressive subcultivation of human embryo fibroblasts (IMR-90). f x p Cell Res 1979; 119: 127-1 43. 30. Paulsson Y, Bywater M, Pfelfer-Ohlsson S et al. Growth factors induce early pre-replicative changes in senescent human fibroblasts. EM80 J 1986; 5: 2157-62. 31. Chang ZF, Chen KY. Regulation of ornithine decarboxylase and other cell cycle-dependent genes during senescence of IMR-90 human diploid fibroblasts. J Biol Chem 1988; 263: 11431-5. 32. Seshadri T, Camplsi J. Repression of c-fos transcription and an altered genetic program in senescent human fibroblasts. Science 1990; 247: 205-9. 33. Rosenberger RF, Gounaris E, Kolettas E. Mechanisms responsible for the limited life span and immortal pheno-
318
34.
35. 36.
37. 38. 39.
Downloaded by [Universität Osnabrueck] at 08:26 16 March 2016
40.
41. 42. 43.
Koli
Keski-Oja
types in cultured mammalian cells. J Theor Biol 1991 ; 148: 383-92. Perelra-Smith OM,Smith JR. Genetic analysis of indefinite division in human cells: identification of four complementation groups. Proc Natl Acad Sci USA 1988; 85: 6042-6. McCarron M, Osborne Y, Story CJ, Dempsey JL, Turner DR, Morley AA. Effect of age on lymphocyte proliferation. Mech Ageing Dev 1987; 41: 21 1-1 8. Cotter TG, Lennon SV, Glynn JG, Martin SJ. Cell death via apoptosis and its relationship to growth, development and differentiation of both tumor and normal cells. Anticancer Res 1990; 10: 1 1 53-60. Arends MJ, Wyllle AH. Apoptosis: mechanisms and roles in pathology. Int Rev Exp Patholl991; 32: 23-54. Kerr JFR, Searle J, Harmon BV, Blshop CJ. Apoptosis. In: Potten CS, ed. Perspectives in mammalian cell death. Oxford: Oxford University Press, 1987: 93-128. McDonald HR, Lees RK. Programmed death of autoreactive thymocytes. Nature 1990;343 624-44. Hameed A, Oisen KJ, Lee MK, Llchtenheld MG, Podack ER. Cytolysis by Ca-permeable transmembrane channels. Pore formation causes extensive DNA degradation and cell lysis. J Exp Med 1989; 169: 765-77. Lockshln RA, Beaulation J. Cell death: questions for histochemists concerning the causes of the various cytological changes. HisfochemJ 1981; 13: 659-66. Stacey NH, Blshop CJ, Halllday JW et al. Apoptosis as a mode of cell death in antibody-dependent lymphocytotoxicity. J Cell Sci 1985; 74: 169-79. Duke RC, Chewenak R, Cohen JJ. Endogenous endonuclease-inducedDNA fragmentation: an early event in cell mediated cytolysis. Proc Natl Acad Sci USA 1983 80: 6361 -5.
44. Chow K-C, Ross WE. Topoisomerase-specific drug sensltivity in relation to cell cycle progression. Mol Cell Biol 1987; 7: 31 19-23. 45..Fesus L, Thomazy V, Falus A. Induction and activation of tissue transglutaminase during programmed cell death. FEBS Lett 1987; 224: 104-8. 46. McCall CA, Cohen JJ. Programmed cell death in terminally differentiating keratinocytes: role of endogenous endonuclease. J lnvest Dermatoll991; 97: 1 1 1-1 4. 47. Whitfield JF. Calcium signals and cancer. Cfit Rev Oncogenesis 1992; 3: 55-90. 48. Szende B, Zalatna A, Schaily AV. Programmed cell death (apoptosis) in pancreatic cancer of hamsters after treatment with analogs of both luteinizing hormone releasing hormone and somatostatin. Proc Natl Acad Sci USA 1989; 8 6 1643-7. 49. Hockenbery D, Nirnez G, Mllllman C, Schrelber RD, Korsmeyer SJ. Bcl-2 is an inner mitochondria1 membrane protein that blocks programmed cell death. Nature 1990; 343: 334-6. 50. ltoh N, Yonehara S, lshll A et al. The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis. Cell 1991 ; 66: 233-4. 51. Trauth BC, Klas C, Peters AMJ. Monoclonal antibodymediated tumor regression by induction of apoptosis. Science 1989; 245: 301 -5. 52. Trump BF, Berezesky IK, Osornlo-Vargas AR. Cell death and the disease process. The role of calcium. In: Bowen ID, Lockshin RA, eds. Cell death in biology and pathology. London: Chapman and Hall, 1981 : 209-42. 53. Wyllle AH. Cell death: a new classification separating apoptosis from necrosis. In: Bowen ID, Lockshin RA, eds. Cell death in biology and pathology, London: Chapman and Hall, 1981: 9-34.
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