Clin Biochem, Vol. 25, pp. 61-75, 1992 Printed in the USA. All rights reserved.

0009-9120192 $5.00 + .00 Copyright © 1992 The Canadian Society of Clinical Chemists.

The Biochemistry of Mammalian Senescence JAMES Institute of Medical

Science,

L. K I R K L A N D

University

Senescence is a process which, until quite recently, has been the subject of little scientific investigation. Even the word "senescence" is difficult to define, and complex methodological pitfalls have impeded progress. In the past few years, there have been exciting advances in understanding the physiological, cell biological, biochemical, and molecular biological nature of senescence. Changes in membrane function, protein synthesis, DNA structure (including glycosylation, altered tertiary structure, free-radical effects, and loss of telomeric DNA), and changes in gene regulation with age are reviewed. Recent work on changes in responses to transcriptional regulatory proteins and cellular senescence factors, some of which have been identified, is particularly promising and leads to the conclusion that senescence, at least in part, is a programmed process. Despite these advances, the fundamental cause of senescence remains elusive but might, as in the case of other biological processes which are phylogenetically widespread, turn out to be quite simple, and perhaps, even modifiable.

KEY WORDS: ageing; senescence; development. Introduction

is one of the most fascinating biological processes, yet the study of senescence has Sonlyenescence recently been accepted as an important area of scientific endeavour. The mechanisms of ageing and senescence are proving difficult to understand; indeed, even the terms "ageing" and "senescence" are not easy to define. Currently, there are no clear biochemical markers of these processes. "Ageing" can be considered as equivalent to "development," embracing embryogenesis, maturation, a period of adult vigour, and senescence. Ageing is a time-dependent process which is progressive, universal within a species, and intrinsic (i.e., not a result of disease or environmental influences). Senescence is t h a t p a r t of the ageing process which is associated with an increasing probability of dying as a function of time (1). In other words, survival curves should be rectangular in species which senesce and which are living under close to ideal conditions, and the onset of senescence in such species can be considered to be the age at which sur-

Correspondence: James L. Kirkland, Room 7238, Medical Sciences Building, University of Toronto, Toronto, Ontario, M5S 1A8, Canada. Manuscript received September 6, 1991; revised November 27, 1991; accepted January 17, 1991. CLINICAL BIOCHEMISTRY,VOLUME 25, APRIL 1992

of Toronto,

Toronto,

Ontario,

Canada

vival begins to decrease rapidly (Figure 1). There is little clear evidence that senescence p e r s e causes death, at least in placental mammals (1,2). Hence, senescence can be defined as that process which predisposes to an increasing probability of dying from diseases or accidents. In order to study senescence, the effects of accidents and diseases have to be separated from those of senescence itself (3). M a m m a l i a n models o f s e n e s c e n c e

Not all species senesce; indeed, there are even some vertebrate species such as sharks and sturgeons which m a y not senesce (4). All placental mammals studied to date appear to undergo senescence (1). Rodents have most often been used in studies of mammalian senescence (5). Many studies in which rodents and other species have been used as a model system of ageing are difficult to interpret because of methodological issues (6). These issues include: (1) ensuring that senescence, rather than maturation, is studied; (2) separating effects caused by accidents, diseases, and other environmental processes from t h o s e c a u s e d b y s e n e s c e n c e ; (3) d e t e r m i n i n g whether a change is directly caused by senescence or is a secondary result of age-related changes in hormonal milieu, activity, diet, or other homeostatic functions; (4) ensuring that animals are nearly identical in all respects except for their ages; and (5) ascertaining whether an apparent ageing change results from a change in the variable of interest or from an age-dependent change in the frame of reference used. In order to study senescence in a species, it is important to understand the life cycle of the species and to compare middle-aged to old animals. There are a number of studies in which conclusions about senescence have been inferred from comparisons between very young and old animals, or even between young and middle-aged animals, in which maturational rather than senescent changes m a y have been observed (reviewed in ref. 6). Effects resulting from senescence itself are difficult to separate from effects of accidents, disease, lifelong sun exposure, exposure to toxins, mechanical wear and tear, gravity, and other environmental influences. These environmental influences, along with true senescent or developmental changes, contribute to the senescent phenotype. However, mea61

KIRKLAND

Time

Specific Age

Figure 1 -- Survival curves of an itsroparous species.Animals living under natural conditions die at a constant rats from disease and accidents (A). As conditions improve, and early deaths from accidents and diseases are prevented, survival curves become more rectangular (B-D). Senescence can be defined as that part of the lifespan showing an increase in mortality in a population maintained under closeto ideal conditions (arrow on curve D). Note that all of the survival curves cross the time axis at the same point (specificage), which is the theoretical upper limit of the species'lifespan.

sures can be taken to ensure that cohorts of animals at different ages have had similar environmental exposures. For example, effects of disease can be reduced by selecting rats born by Caesarian section and raised under double-barrier conditions and excluding rats if antibody profiles suggest that the animals have experienced illnesses which could confound results or if autopsy shows disease. Furthermore, strains of animals selected for ageing studies should be free, as far as possible, of genetic conditions likely to confound the results. While senescent processes may themselves be genetic, they should be universal within a species and not sporadic, as in the case of genetic diseases. Some biochemical and physiological alterations in ageing rats m a y result from changes in activity, diet, hormonal milieu, and other variables. For example, accelerated postmenopausal bone loss is most likely an effect of hormonal changes which occur after the menopause (7). The accelerated bone loss is secondary to another process which is more directly associated with ageing. One could not hope to learn much about the cause or nature of senescence itself by studying the mechanism of the accelerated bone loss, although the latter is an important subject for research in its o w n right. In order to isolate changes caused by senescence from effects of changing homeostatic milieu, it is necessary to control the milieu so that it is similar in subjects of all ages studied. O n e w a y to achieve this is to take cells or organs from animals or h u m a n subjects of different ages

62

and to study them in culture (8,9). This approach also reduces the confounding effects of disease and other environmental factors. In ageing studies, animals should be identical to each other in all respects except for their ages. In addition to approximating identity of environmental conditions and internal milieu among age groups, genetic identity should be ensured insofar as possible. This can be achieved by using animals as their own controls (longitudinal studies) or by studying d i f f e r e n t age cohorts of i n b r e d s t r a i n s (crosssectional studies). The longitudinal approach has many advantages. Individuals are used as their own controls, and this helps to factor out the effects of genetic influences. Results are not affected by "selective mortality" (younger cohorts include some individuals who are likely to die before achieving the age of the older cohorts, so that the older cohorts represent a selected population of fit individuals). "Cohort effects" caused by exposure of one age cohort but not others to environmental influences (e.g., an epidemic) are reduced. Longitudinal studies have a number of disadvantages. Repeated measurements, especially if these involve any stress to the animals (e.g., removal of tissue for study at intervals), can cause effects that may be misleading. Longitudinal studies are more expensive and time consuming than cross-sectional studies. Cross-sectional ageing studies can yield reliable information if inbred strains are used, and these are often more practical than longitudinal studies. Changes in a physiological or biochemical variable expressed as a proportion can result from agedependent changes in the denominator (e.g., see ref. 10). For example, the fat cell receptor number can be expressed as a function of wet tissue weight, triacylglycerol content, total protein content, cell number, or cell surface area. F a t cell diameter decreases between middle and old age in the epididymal pads of Fischer 344 rats (11). Therefore, the age-related decrease in cell volume (triacylglycerol content) is greater than the decrease in cell surface area, which in turn is greater than the decrease in cell number. Even if receptor number per cell remained constant with age, apparent receptor concentration would increase ifrecepter numbers were expressed as a function of cell surface area or triacylglycerol content. This denominator effect greatly complicates interpretation of changes with age in biochemical or physiological p a r a m e t e r s in adipose t i s s u e (6). Model systems chosen for ageing studies must be free of this phenomenon or results m u s t be presented as a function of each denominator which m a y be of physiological importance. Cell biology of senescence REPLICATIVE ARREST

In 1961, Hayflick and Moorehead (12) reported that cultured fetal h u m a n fibroblasts can undergo only a limited number of population doublings beCLINICAL BIOCHEMISTRY,VOLUME 25, APRIL 1992

BIOCHEMISTRY OF SENESCENCE fore arrest of cellular replication occurs. In successive subcultures, the time to reach confluence increases (i.e.,population-doubling time increases) until, eventually, cultures fail to achieve confluence, although the cells remain viable (13). Eventually, cell viability also decreases and the "Hayflick limit" is reached. This phenomenon has now been observed in a variety of celltypes from a number of vertebrate species (14). So far as is known, the only euploid mammalian cells in which growth arrest does not occur are components of the immune system (15). These cells, in which genetic rearrangements are important in differentiation, m a y have different mechanisms controlling replication and differentiation from other cell types. The number of population doublings reached before growth arrest correlates with donor age. Hum a n adult lung fibroblasts are not capable of achieving as m a n y population doublings as fetal lung fibroblasts (13). The proliferative potential of cloned rat preadipocytes and skeletal muscle satellitecells decreases with increasing donor age (16,17). The replicative potential of h u m a n skin, skeletal muscle, and bone marrow fibroblasts grown under both mass culture and clonal conditions is inversely related to donor age in h u m a n subjects from the frst to ninth decades of age (8,9,18).Furthermore, the number of population doublings achieved correlates with the m a x i m u m lifespan of the species. Fetal mouse fibroblasts survive eight population doublings before the growth arrest which precedes transformation occurs, while fetal h u m a n fibroblasts last for 50 or more doublings before growth arrest (19). The maxi m u m lifespan of most mouse strains is 36 months and of humans is 120 years (14). Thus, the limited population-doubling potential of cultured cells m a y be related in some way to the process of senescence. Indeed, the process has been termed "cellular senescence" (12). A few factors other than donor age and species m a x i m u m lifespan affect the number of population doublings achieved in serial cultures. Cultured fibroblasts from patients with Werner's syndrome and diabetes mellitus achieve fewer population doublings before growth arrest than fibroblasts from healthy control subjects (18,20). Replicative potential varies among different tissues from the same animals. H u m a n lung fbroblasts undergo more population doublings than skin fibroblasts, skin fibroblasts more than skeletal muscle fibroblasts, and skeletal muscle fibroblasts more than bone marrow fibroblasts (18,21). These differences among tissues might reflect the number of divisions that cells in those tissues had undergone during embryogenesis and subsequent stages of development before harvesting. Components of the culture medium have been reported to affect the number of population doublings achieved. Epidermal growth factor (EGF) extends the number of population doublings achieved by newborn h u m a n epidermal cells in culture (22). E G F does not have this effecton h u m a n skin or lung CLINICALBIOCHEMISTRY,VOLUME25, APRIL 1992

fibroblasts or embryonic human flbroblasts (23,24). Possibly, EGF acts by delaying differentiation of epidermal cells. Use of serum-free culture media can result in greatly increased numbers of passages before growth arrest occurs (25,26). In fact, mouse embryo cells can be cultured in serum-free medium without growth arrest or the appearance of gross chromosomal aberrations for many months (26). However, it is possible that a transforming event occurs in rodent cell cultures which results in unlimited growth potential but which does not result in development of morphological features of transformation or in tumorigenicity. Such events have been observed in mouse fibroblasts (27). Hydrocortisone exposure may increase the population-doubling capacity of mass-cultured fibroblasts under certain conditions (28). This may be due to an increase in the proportion of cells which achieve maximum replications rather than an increase in the maximum number of divisions (21,29). These observations suggest that the Hayflick effect is not absolute, but they are compatible with the hypothesis that limited population-doubling potential is causally linked to the process of senescence. A number of factors do not affect population doubling potential. Culture at room temperature instead of 37 °C prolongs the total duration of cell survival, but does not increase the number of population doublings achieved before growth arrest occurs (14). Vitamin E has no effect (30). Chromosomal composition does not appear to have an effect on the population-doubling capacity of human fibroblasts (31). Also, even though females live longer than males in many mammalian species (1), gender has no effect on cell-replicative capacity (32). Replicative arrest of late passage cells appears to occur during the gap 1 (G 1) phase of the cell cycle (33-35). When late passage fibroblasts are fused to cells of the T98G line, T98G nuclei in the synthesis (S) phase at the time of fusion continue to synthesize DNA (assessed by 3H thymidine exposure and autoradiography), while T98G nuclei in G1 are unable to synthesize DNA (33). When early- and late-passage cells are fused, DNA synthesis is inhibited in earlypassage nuclei in the G1 phase but not in nuclei in the S phase (35). When early-passage cells are in G1, but are within 3 h of entering the S phase of the cell cycle, DNA synthesis can still occur aider fusion to late-passage cells (34). Thus, replicative arrest in late-passage cells probably occurs at a point during G1 within 3 h of the G1-S interface. The replicative limit is probably rarely achieved by cells in vivo. However, a number of metabolic changes occur in serially passaged cells well before growth arrest (14). These changes in function could be more important in the genesis of the senescent phenotype than growth arrest itself. In human diploid fibroblasts at late passage, these functional changes include increased lipid content, lipid synthesis, glycogen content, protein content, RNA content and turnover, and activities of proteolytic and other catabolic enzymes (reviewed in ref. 14). Possi63

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bly, these functional changes are linked to the processes which result in decreased cellular replicative capacity in late-passage cells. Perhaps the processes which result in changing function in nondividing cells (e.g., neurons) with ageing are also related to the processes which cause the Hayflick effect (14). However, some of these functional changes (e.g., increased protein and RNA content) have not been observed in fibroblasts freshly isolated from old donors (8). The Hayflick phenomenon has been observed in situations other than serial passage of cultured cells (reviewed in ref. 36). Growth potential decreases in mouse m a m m a r y tissue which has been serially transplanted into isogenic hosts (37). Reinitiation of growth in the transplanted tissue occurs when cholera toxin-releasing plastic implants are placed near the transplanted tissue in vivo (38). This suggests that mitotic dysfunction in serially transplanted tissue may not result from generalized cellular deterioration, but rather from a specific change in cell regulation. Outgrowth of cells from tissue explants occurs more slowly, and less extensively, in tissues from old than from young donors (39). The average size of colonies resulting from division of individual human skin fibroblasts in culture becomes smaller with increasing donor age; the size of colonies correlates with the number of population doublings achieved in serial culture (9). Additionally, the ability of aortic smooth muscle cells to attach and replicate after plating at low culture density (cloning efficiency) decreases progressively with advancing donor age in mice as does cloning efficiency of rat preadipocytes (17,40). Perhaps age-related changes in cloning efficiencies are related, in some way, to the Hayflick phenomenon. DIFFERENTIATION

The Hayflick effect could be a manifestation of differentiation rather than of senescence (41). For example, the differentiation of 0-2A oligodendrocyte progenitor cells into oligodendrocytes in vitro appears to be controlled by a developmental clock which counts cell divisions (42). In conditions which promote their differentiation, cloned 0-2A progenitors divide from one to eight times before they stop replicating and differentiate. Sister cells undergo the same number of divisions before differentiating. At least in the 0-2A cell line, cell division counting could explain the timing of differentiation. Both differentiation and the Hayflick effect could be related to a cell-division counting process. However, the distinction between "differentiation to death" and ageing could be semantic (14). Ageing and differentiation could be related to the same fundamental mechanism, and the Hayflick effect may be related to both senescence and terminal differentiation. Terminal differentiation does not appear to account for growth arrest in serially passaged adrenocortical cells. When individual adrenocortical cells are cultured and clonal capacities for replication and 64

differentiation are analyzed, loss of replicative potential due to late passage arrest does not correlate with expression of a differentiation-dependent gene (43,44). TRANSFORMATION AND CELLULAR SENESCENCE

After transformation, cells acquire the capacity to survive through an unlimited number of subcultures. However, cell hybrids made by fusing latepassage fibroblasts to transformed cells have limited replicative potentials, while hybrids formed by fusion of certain types of transformed cells to one another can replicate indefinitely (45). Ribosomal RNA (rRNA) synthesis is reduced in nuclei of latepassage h u m a n fibroblasts (46). When rRNA synthesis is measured in each nucleus of the hybrids by autoradiography, inhibition of RNA synthesis is observed in nuclei of early-passage cells fused to latepassage cells while unchanged RNA synthesis is observed in nuclei of early-passage cells fused to other early-passage cells. Hence, dominant intracellular factors may be responsible for growth arrest in latepassage cells. Indeed, when late-passage cells are enucleated and the resulting cytoplasts are fused to early-passage cells, replication of the resulting hybrids is blocked (47). Cycloheximide or puromycin treatment of the late-passage cells eliminates the potential of cytoplasts made from them to block replication. When late-passage cells are treated with cycloheximide, enucleated, and then allowed to recover, the cytoplasts regain their capacity to block replication when fused to early-passage cells. Messenger RNA (mRNA) isolated from late passage cells causes growth arrest after microinjection into early-passage cells, while m R N A isolated under identical conditions from early passage cells does not cause growth arrest (48,49). Therefore, "cellular senescence" proteins m a y be responsible for the Hayflick phenomenon. Several different cellular senescence factors may exist. Fusion of transformed cells of any particular line to each other results in immortal hybrids. Fusion of cells of certain transformed lines to those of other transformed lines also results in immortal hybrids in some cases, but not in others (49). It has been suggested that different cell lines belong to one of several complementation groups, each of which has been transformed by a different mechanism and each of which is suppressible by a different cellular senescence factor. Hence, fusion of cells of a line belonging to one complementation group to cells of a different line belonging to the same group results in an immortal hybrid line, while fusion of a cell line from one group to a line from another group results in cells subject to eventual replicative arrest. Fibronectin may be one of the cellular senescence factors (50); another may be an inhibitor of phosphorylation of the retinoblastoma gene product, P l l 0 Rb. Unphosphorylated P l 1 0 Rb inhibits entry into the S phase of the cell cycle, and levels of unphosphorylated P l l 0 Rb are high in late-passage cultured fibroCLINICAL BIOCHEMISTRY, VOLUME 25, APRIL 1992

BIOCHEMISTRY OF SENESCENCE

blasts (51). Introduction of SV40 T antigen, adenovirus E1A, or H P V E7, which preferentially bind to and possibly inactivate unphosphorylated P l l 0 Rb, induces division of late-passage fibroblasts. Serum, which s t i m u l a t e s p h o s p h o r y l a t i o n of P l 1 0 Rb in e a r l y - p a s s a g e q u i e s c e n t (serum-deprived) fibroblasts, does not stimulate phosphorylation in latepassage fibroblasts. An inhibitor of phosphorylation of P l l 0 Rb m a y be present in late-passage fibroblasts, or there m a y be failure of serum to induce production of activators of phosphorylation. Another cellular senescence factor could cause repression of transcription of c-fos, the product of which is required for cell division in fibroblasts. Repression of c-fos is specific, since serum does not induce expression of c-fos in l a t e - p a s s a g e h u m a n fetal l u n g f i b r o b l a s t s , whereas it does in early-passage cells. Serum enhances c-myc and c-Ha-ras expression in both earlyand late-passage cells (52,53). A negatively acting P l l 0 RD response element is located in the 5' promoter region of the c-fos gene, and changes in P l l 0 Rb function and c-fos expression in late-passage cells could be causally related (54). Repression of c-fos transcription is, however, unlikely to be the only mechanism through which late-passage arrest occurs, because introduction of a c-fos expression vector, which results in increased nuclear levels of fos protein, does not induce replication of latepassage fibroblasts (54). Cellular senescence factors could be tissue specific. Late-passage h u m a n endothelial cells contain high levels of interleukin-la (55). Introduction of an antisense oligomer which binds to interleukin-la transcripts delays replicative arrest in endothelial cells. However, since replicative arrest is only delayed and not eliminated, other senescence factors may also mediate replicative arrest in these cells. Also, since i n t e r l e u k i n - l a is mitogenic in fibroblasts, it may be a tissue-specific cellular senescence factor. In humans, at least one cellular senescence factor may be encoded by a gene on chromosome 1 (56). Most of the hybrids made between euploid h u m a n flbroblasts and a transformed, immortalized hamster cell line have a capacity for cell replication which is similar to that of h u m a n fibroblasts. This confirms the notion that dominant cellular senescence factors are present in the hybrids. Some of the hybrids do give rise to immortalized lines, and all lack h u m a n chromosome 1. Additionally, introduction of h u m a n chromosome 1 into immortalized hamster cells by microcell fusion results in hybrids with limited replicative potential. Hence, h u m a n chromosome 1 m a y produce a cellular senescence factor able to overcome the alteration in the hamster cell line which results in its unlimited replicative potential. Recently, using the same approach as in the studies of chromosome 1, h u m a n chromosome 4 has been found to confer senescence upon three different human-transformed cell lines (57). The three lines all belong to the same complementation group. In the hamster, a gene involved in cellular senesCLINICAL BIOCHEMISTRY, VOLUME 25, APRIL 1992

cence appears to be on the long arm of the X chromosome (58). W h e n microcells containing a normal Chinese hamster X q chromosomal fragment are fused to cells of a nickel-transformed hamster line, the hybrids lose their capacity to replicate indefinitely. Hence, another m a m m a l i a n cellular senescence factor m a y be produced or influenced by the product of a gene on the X chromosome. Different transformed cell types appear to be immortalized through different mechanisms, and it would not be surprising if several different chromosomes in each mammalian species carry cellular senescence genes, each specificfor a different complementation group. These cellular senescence genes could be important as a defense against cancer, giving relevance to the existence of redundant systems. Biochemical changes observed in senescent m a m m a l s MEMBRANE

CHANGES

Several changes in membrane characteristics occur during senescence. Whether observed changes are caused by ageing itselfor by disease processes, other environmental influences, or methodological artifacts, depends upon the model system and the experimental design used. Changes in the carbohydrate moieties of m e m brane glycoproteins m a y occur with age (59). Latepassage h u m a n fibroblasts incorporate 3H-fucose into glycoproteins more extensively than do earlypassage fibroblasts, while incorporation of 3Hglycosamine does not change. Changes in the carbohydrate moieties of membrane glycoproteins and glycolipids could affect membrane function. M e m brane fluiditydecreases during senescence (60). Decreased fluidityis related to increases in membrane cholesterol and saturated phospholipid content. There is an age-related decrease in cholesterolturnover and membrane phospholipid synthesis. These changes in membrane fluiditycan be reversed by in vivo or in vitro treatment with unsaturated lipid preparations (61). Membrane fluidity or phospholipid composition affects receptor-mediated transfer of signals across cell membranes (62-65). This results in decreased sensitivity to various hormones with ageing. Sensitivity can be partially restored by treatment with unsaturated lipids. Decreased m e m b r a n e fluidity also results in increased osmotic fragility of cells from old animals. The numbers and affinitiesof receptors for various hormones increase, remain the same, or decrease with age in differenttissues. For example, in the heart there m a y be a decrease in ~-receptor number, in the liver there is an increase in ~-receptor number, and in white blood cells the affinity of ~-receptors decreases but receptor number does not change (66,67). To date, no changes in the primary structure of receptors with ageing have been reported, although the proportions of various receptor isotypes m a y change. 65

KIRKLAND As erythrocytes age, a senescent cell antigen appears which is important in physiological removal of old erythrocytes from the circulation (68). Senescent cell antigen is a product of the breakdown of the membrane-spanning portion of band 3 glycoprotein, which m a y have a functional relationship to the erythrocyte glucose transporter (69). Several cell membrane- or extracellular-matrixassociated antigens have been found in late but not early-passage fibroblasts (70). These antigens are present on late-passage h u m a n foreskin, lung, and skin fibroblasts, but are not expressed by earlypassage fibroblasts,serum-deprived quiescent earlypassage fibroblasts, or immortalized transformed h u m a n cells. The three antigenic determinants all turn out to be located on the fibronectin molecule. Antibodies against the three determinants react against intact late-passage but not early-passage cells, although they do react with fibronectin extracted from early-passage cells. Hence, late-passage cell surface fibronectin m a y present an epitope distinct from fibronectin on early-passage cells due to changes in cell surface fibronectin conformation. Fibronectin expression is upregulated 10-fold in late compared with early-passage fibroblasts (71).

opment. For example, the isozyme pattern of glyceraldehyde-3-phosphate dehydrogenase in rat muscle changes with age (79,80). There is no evidence of any alteration in the primary structures of these isozymes. The t e r t i a r y structure of some proteins may change with age. The activity of phosphoglycerate kinase in rat muscle decreases with age and its heat stability changes (81). However, after exposure to denaturing conditions and subsequent reactivation by dilution into a large excess of denaturant-free buffer, the enzyme from old rats has the same activity as that of enzyme derived from young animals. The amino acid sequence ofphosphoglycerate kinase is the same in young as in old animals. This indicates that a change occurs during ageing in the postsynthetic modifications which affect the tertiary structure of phosphoglycerate kinase. Thus, there is little indication from studies of protein characteristics that a change occurs in the fidelity of gene transcription with age, but there appear to be alterations in gene regulation and changes in the nature of postsynthetic modifications of proteins resulting in alterations in tertiary structure and, possibly, in patterns of glycosylation.

CHANGES IN EXTRACELLULAR PROTEINS WITH AGE

CHANGES IN

Slowly turning-over extracellular proteins, such as collagen or crystallin, may undergo gradual changes, which affect their function and contribute to the senescent phenotype. For example, the nonenzyme-dependent Maillard reaction of reducing sugars with amino acids to form stable brown pigments may contribute to crosslinking of collagen and other macromolecules (72). The crosslinking affects elasticity and other properties of the macromolecules. However, ageing does not appear to be related to the extent of glycosylation of proteins which have half-lives of up to several months, such as hemoglobin, in healthy, nondiabetic individuals (73). The Maillard reaction may contribute to changes in the properties of tendons, the lens, and other tissues which contain slowly turning-over proteins, but it may not be important in the case of most intracellular proteins. However, this reaction could contribute to changes in DNA function with age (74).

Little definitive information is available about effects of age, if any, on RNA characteristics. Studies of changes in RNA polymerase II with age have yielded inconsistent results (82-85). Rates of RNA splicing, of addition of the poly (A +) tail, and of energy-dependent transport of poly (A +) RNA from the nucleus to the cytoplasm may be reduced with age (86), but there are no changes in the lengths of poly (A +) tails or in posttranscriptional capping (87). The translational activity of mRNA isolated from rat hepatocytes in a reticulocyte lysate cell-free system appears to be unaltered with age (87). Reduced degradation of albumin mRNA with age has been observed, but effects of altered hormonal milieu, activity, nutrition, and other variables could be the cause of this, rather than senescence (76).

CHANGES IN ENZYMES AND OTHER INTRACELLULAR PROTEINS WITH AGE

D N A structure

Protein synthetic rates decrease with age in many, but not all, mammalian species and tissues (75,76). In the rat heart, for example, protein synthesis does not appear to be affected by age (77). Also, synthesis of specifc proteins m a y increase [e.g.,tyrosine hydroxylase (78)],decrease, or remain constant with age. Altered protein degradation rates can contribute to increased or decreased steady state levels of various proteins with age. Also, changes m a y occur in the relative activities of various isozymes in senescence, as in other phases of devel66

RNA FUNCTION WITH AGE

CHANGES IN DNA STRUCTURE, REPLICATION, REPAIR, AND FUNCTION DURING AGEING

Putative age-related changes in D N A replication, repair, and transcription could be related to changes in D N A structure. Effects of age on the primary and tertiary structure of D N A have been studied extensively. The age-related changes in primary structure which have been reported include an increase in the frequency of single-strand breaks, decrease in D N A methylation, and loss of telomeric D N A sequences (88-93). A n age-related change in tertiary structure, loss of negative supercoiling, m a y also occur (94). Evidence for increased frequency of single-strand CLINICALBIOCHEMISTRY,VOLUME25, APRIL 1992

BIOCHEMISTRY OF SENESCENCE breaks has come from studies employing sucrose density gradient centrifugation of D N A under alkaline conditions, from deoxynucleotide-incorporation experiments, and from studies of D N A sensitivityto $1 nuclease using D N A from animals of various ages or cells at various in vitro population-doubling levels (88-92). Single-strand breaks can delay D N A synthesis by reducing negative supercoiling, but have little effect on protein structure (94-95). Whether increases in single-strand breaks actually occur has been questioned, and it is possible that D N A isolation methods or changes in nuclease activities with age result in an apparent increase in single-strand breaks (96). Decreases in D N A methylation occur with serial passage of diploid fibroblasts (97). Methylcytosine content decreases in tissues from ageing cattle and salmon, but not from humans (97). Demethylation m a y enhance the production of at least one cellular senescence factor (58). W h e n normal hamster X-chromosomes are transferred into cells of an immortalized nickel-transformed hamster line, the hybrids reacquire the capacity to undergo replicative arrest. Normal X-chromosomes from late-passage cells are less effective in inducing replicative arrest in the hybrids than are X-chromosomes from early passage cells.Treatment of X-chromosomes from the late-passage cells with 5-azacytidine, which induces demethylation, enhances the capacity of the X-chromosomes to cause replicative arrest. Hence, changes in methylation patterns m a y have a role in the appearance of changes associated with ageing. However, changes in methylation patterns are epigenetic and must themselves be caused by some more fundamental process. Thus, it is not clear how important they are in the causation of senescence, or as seems more likely, whether these changes are effects of a more fundamental process and are a link in the causal chain of events which result in phenotypic manifestations of senescence. At the least, changes in methylation patterns m a y prove to be useful as markers of ageing. Loss of telomeric D N A m a y contribute to senescence (93,98).Telomeres are segments of D N A at the termini of chromosomes and are comprised of stretches of species-specific, G-rich repeats (99). A ribonucleoprotein enzyme, telomerase, synthesizes telomeric D N A , instead of the D N A polymerases involved in semiconservative D N A synthesis (100). Functions of telomeres could include prevention of chromosomal instability, fragmentation, and rearrangement, and prevention of the loss of terminal D N A sequences which occurs during semiconservative synthesis, because D N A polymerases leave 5' terminal gaps after each replication round. In yeast and Tetrahymena, mutants have been described which have abnormal telomeres and which have restricted replicative potential likened to senescence (100,101). In serially passaged h u m a n fibroblasts, around 50 bp per telomere of D N A are lost with each celldivision (93). Somatic h u m a n chromosomes have telomeres with around 4 kb of D N A , so CLINICALBIOCHEMISTRY,VOLUME25, APRIL 1992

that most telomeric DNA is lost aRer 40 cell generations. Additionally, human sperm have around 9 kb of telomeric DNA, fetal cells have more telomeric DNA than cells from adults, and length of telomeric DNA in human blood cells is inversely related to age (93,98). Loss of telomeric DNA may, therefore, be a cause of or be caused by senescence. In lower eukaryotes and in transformed human cells, telomere length does not appear to shorten with successive cell divisions, and is maintained by the presence of telomerase (98,99). Failure to replace lost telomeric DNA in somatic human cells could result from reduced activity of telomerase, alterations in the amounts or function of specifc telomere-binding proteins, or other mechanisms. However, direct evidence for any of these mechanisms is lacking. Additionally, telomere length does not correlate well with extent of restriction in replicative capacity ("senescence") in protozoans with telomerase mutations (100). Information about correlations among telomere length, rate of loss of telomeric DNA during successive cell generations, and specific age in different mammalian species is not yet available, nor are observations about effects of manipulating telomere length on cell-replicative potential. It is too early to assume that shortening of telomeres causes senescence, although this is an important topic for further study. Loss of negative supercoiling of DNA has been postulated to occur with ageing (94). Loss of negative supercoiling would have profound effects on DNA replication, repair, and transcription (102). Data from studies of nucleoid sedimentation in sucrose gradients after x-ray exposure or treatment with ethidium bromide are consistent with the hypothesis that negative supercoiling decreases aider serial passage in fibroblasts (94). Lower x-ray doses were required to achieve a minimum sedimentation rate of nucleoids prepared from late-passage than from early-passage cells. Several factors confound the interpretation of these results. Nucleoid preparation involves exposure of DNA to salt concentrations which can affect supercoiling. Nuclease and lysosomal enzyme activities are high in late-passage cells (14). However, studies of nucleoids from liver and brain of maturing mice support the conclusions drawn from the serial passage studies (94). Hence, a reduction in negative supercoiling with ageing could be a step in the causal chain of events which result in the senescent phenotype.

DNA replication Ageing is associated with reduced proliferative potential and decreased D N A replicative capacity in dividing cells.D N A replication also slows during serial passage of h u m a n fibroblasts (103). Early- and late-passage cells undergoing exponential growth were exposed to SH thymidine in 10-rain to 3-h pulses. Cells were spread on slides, D N A was precipitated and fixed, and track lengths and grain densitieswere measured by autoradiography. A 25% re67

KIRKLAND duction in rate of chain elongation was observed in late-passage cells.Since there were no differencesin centre-to-centre distances of the tracks, it appears that there is no change in the distance between D N A replicationinitiationsiteswith passage. From these observations, it was concluded that D N A replicationrates decrease with age, that the decrease is not related to a reduction in the number of D N A replication initiationsites, and that the effect m a y be a result of defective or deficient enzymes or substrates involved in D N A replication with age. Delayed D N A replication has been observed in in vivo studies of young and old animals. At intervals from 5-180 min a i ~ r intravenous injection of 3Hthymidine, 6- and 24-month-old rats were killed, and nuclei from spleen cells were isolated and disrupted on a continuous alkaline sucrose gradient which contained a lysing solution (104). Changes with age in rates of chain growth were estimated from changes in the position of labelled peaks along the gradient and changes in total 3H-thymidine uptake. Labelled DNA from old rats shifted to the bottom of the gradient more slowly than that from young animals. Total uptake of label was reduced in old rats. From these results, it appears that the rate of DNA synthesis is slower in old rats. DNA ligase activity could be reduced, resulting in the persistence of small fragments of DNA in old animals. Additional evidence for an effect of ageing on DNA replication came from a study of mice from an ageing colony for which growth curves and other colony characteristics were well defined (105). Sixty to 70% of the animals' livers were excised and, at various intervals after surgery, high doses of 3Hthymidine were injected. One hour later, the regenerating liverswere removed. Animals with evidence of disease were excluded, and the distribution of liver cell types (parenchyrnal and non parenchymal) was similar in animals of all ages. ~H-thymidine/ D N A levels were measured in fragments of regenerating livers. M a x i m u m levels occurred in 6-month-old mice 48 h after partial liver resection and in 28-month-old animals 70 h afterresection.To avoid problems associated with changes in intracellular thymidylate levels with age, the proportion of cells which took up label at various intervals after partial resection was determined. Peak proportions were observed in young mice 24-48 h before peaks in old animals in both parenchymal and nonparenchymal liver cells. Hence, D N A replication after partial liver resection is delayed in very old compared to immature mice. However, it is not clear from these data whether one of the m a n y factors, such as glucagon levels,E G F effects,or insulin concentration, which mediate the timing of initiationof cell replication after liver resection are altered, or whether the process of D N A replication itself is slowed in old age. Replicative D N A synthesis has been examined in hepatocytes cultured from very young (2-3-monthold) and senescent (28-30-month-old) rats (106). Upon exposure to EGF, these cells divide. There

68

were neither differences in the affinity nor number of EGF receptors between the young and old rats; however, DNA synthesis assayed by measuring 3Hthymidine, incorporation into ~)NA was reduced in cultures from old rats. Hence, postreceptor events appear to account for the reduced DNA synthetic response to EGF in hepatocytes. The changes observed in whole-tissue DNA synthetic rates during senescence could be explained by an increasing proportion of individual cells being arrested at the G1-S interface or by a decreased rate of DNA synthesis during the S phase within each cell. The observation that late-passage cells become blocked at the G1-S interface in serial culture experiments supports the conclusion that reduced tissue DNA synthetic rates are related to an increasing probability of late G1 arrest during senescence.

DNA repair Many of the changes prevalent in senescent animals occur in fixed, nonmitotic, or slowly replicating cells such as neurons or T lymphocytes (1). Changes in unscheduled DNA repair capacity could be associated with the effects of ageing in nonmitotic tissues. Conclusions from early studies indicate that late-passage fibroblasts have a lower rate of unscheduled DNA repair synthesis after UV radiation than early-passage cells, and that muscle cells from old rats are not capable of repairing X-ray-induced DNA breaks as efficiently as cells from young animals (107,108). There may be a relationship between UV-induced unscheduled DNA synthesis and maximum lifespan of the species (109). Seven species whose maximum lifespans ranged from 1.5 years (shrew) to 120 years (human) were studied. Skin biopsies from animals which had achieved 5% of their maximum lifespan were obtained, fibroblast cultures were prepared, replication was inhibited with hydroxyurea, cultures were exposed to sufficient UV radiation to induce a maximum unscheduled DNA repair response, and 3H-thymidine uptake was measured. Rates of repair synthesis correlated with species maximum lifespan. However, in a later study of 34 species, no correlation was found between species maximum lifespan and rate of unscheduled repair synthesis (110). There were serious methodological problems with the latter study (e.g., some cultures were from skin and others from lung, and no allowance was made for donor age); nevertheless, the results cast some doubt upon the results of the first study. Additionally, the physiological significance of capacity for repair of UV-induced lesions is not certain, since this repair occurs through mechanisms different from those responsible for the repair of other types of DNA damage (111,112). Also, changes in UV damage repair synthesis do not necessarily correlate with changes in DNA synthetic capacity (106). In cultures ofhepatocytes from old rats, DNA synthetic capacity is reduced compared to young animals, while UV and N-nitroso-N-methylurea-induced unCLINICALBIOCHEMISTRY,VOLUME25, APRIL 1992

BIOCHEMISTRY OF SENESCENCE

scheduled repair synthesis is unchanged. It is not clear whether physiologically important changes in D N A repair capacity occur with ageing.

DNA polymerase If ageing decreases the rate of D N A synthesis (as opposed to the probability of initiation of replication), the decrease could be related to alterations in D N A polymerase activity, other enzymes involved in replication (e.g., D N A ligase, unwinding enzymes, etc.), substrate availability, enzyme cofacters, histone binding, or the structure of D N A itself. If ageing decreases the probability of initiation of replication, the decrease could be caused by alterations in the control mechanisms which transduce extracellular stimuli to initiatereplication,or by altered function of the intracellular processes which control time intervals between cell divisions. A number of investigators have examined the effects of age on D N A polymerase activity (105,113124). There are at least three types and several subtypes of D N A polymerase in mammalian cells(102). D N A polymerase-a appears to be associated primarily with cell replication, polymerase-~ with repair, and polymerase-~/ with mitochondrial function. A number of investigators have reported decreased polymerase-a activity and fidelity (assayed by measuring misincorporation of labelled nucleotides into D N A templates) and decreased polymerase-~ activity with age (105,116,119-123,125-127). Changes in the physical properties, activities,and fidelity of polymerase isozymes with age could reflecterrors in enzyme synthesis, increases in protease activities, or changes in isozyme production patterns in ageing cells (105,116,120,121). Activators or inhibitors of polymerase activity did not appear to account for these age-related changes, nor did changes in the activity of terminal deoxynucleotidyl transferase, which can affect the fidelity of D N A synthesis (119,120). Decreased or altered polymerase-a activity with age m a y be related to reduction in the proliferative potential of ageing cells,and m a y even contribute to this reduction. The observation that D N A polymerase from old cells catalyses D N A replication with less fidelity than enzyme from young cells was of considerable interest. A positive feedback cycle could be envisaged which involves faulty D N A replication resulting in D N A sequence errors which, in turn, could result in production of faulty enzymes in general, and faulty D N A polymerase in particular (116,120). Attractive as this hypothesis was, it is no longer tenable (122,125,126). The fidelitiesof D N A polymerase isozymes depend on their specific activities. Since the activities of polymerases are low in the ageing systems where poor fidelityhas been observed, the reduction in fidelityassociated with ageing is probably a consequence of low enzyme activities.Purified polymerase-a and -~ from liver tissue of old mice transcribe D N A with the same fidelityas enzymes purified from young mice and polymerCLINICAL BIOCHEMISTRY, VOLUME 25, APRIL 1992

ase-[~ from brains of aged mice also appears to have the same fidelity as that from young mice (125,126). Furthermore, at least two forms of polymerase-a appear to exist in humans: a high-affinity form and a low-affinity form (127). The former is the predomin a n t type in fetal tissue, while the low-affinity form increases in activity relative to the high-affinity form with increasing donor age. The low-affinity form can be converted to the high-affinity form by t r e a t m e n t with phosphatidylinositol, ATP, and phosphatidylinositol kinase. Hence, reduced activity (and, therefore, fidelity) of polymerase-a with increasing age may result from loss of an enzyme activator molecule, and changes in polymerase-a function appear to result from a more fundamental process linked with senescence. OTHER

FACTORS W H I C H

MAY

AFFECT

DNA

SYNTHESIS

Changes with age in properties of D N A ligase, unwinding enzymes, and other enzymes associated with D N A synthesis have not been reported. The activity and physical characteristics of terminal deoxyribonucleotidyl transferase appear to change during maturation, but no changes have been found during senescence (119,120,128). A n u m b e r of changes in histones are reported to occur during ageing, including altered ease of extraction from DNA, decreased histone acetylation (which could be secondary to changes in cytoplasmic acetyl C o A levels), increased histone H1 o relative to H1 levels, altered ratio of methionine-containing to methionine-free H1 o, and changes in histone repeat length (129-133). Altered histone-masking of D N A m a y occur with ageing (134). However, these changes in histone characteristics are likely to be consequences of other events (including changes in histone synthesis) which occur with ageing (129,135). There is littledoubt that net rates of cell proliferation decrease during senescence. Decreased D N A synthetic rates or a decreased probability of initiation of replication in individual cells could account for this. While there are tenuous assumptions and flaws in the conclusions reached in m a n y of the reports of investigations of effects of age on D N A synthesis and repair, the results are generally compatible with the conclusion that there are changes in D N A synthetic capacity with age. However, alterations in D N A structure, in synthetic rates, or in the probability of initiation of synthesis with age could be epiphenomena, or an intermediate link in the causal chain of events which result in the senescent phenotype. The fundamental question is: W h a t starts the causal chain of events which result in changes in gene transcription, D N A replication, and cell proliferation which, in turn, result in the senescent phenotype? Hypotheses concerning the cause of senescence There are a number of hypotheses concerning the initiating event which culminates in senescence, and they are not all mutually exclusive. 69

KIRKLAND WASTE PRODUCT THEORY

According to this theory, accumulation of nondegradable metabolic waste products results in compromised cell function (136). Lipofuscin has been considered to be such a waste product. It is composed of lipids, carbohydrates, and proteins and is stored in irregular, granular, fluorescent inclusion bodies in cells of senescent organisms, particularly in postmitotic cell types such as neurons or smooth muscle cells. Up to 30% of the volume of such cells from old animals can be comprised of lipofuscin. Lipofuscin is metabolically active and its amount in neurons can be reduced by various experimental manipulations (137,138). Additionally, while lipofuscin accumulates in cells prevented from dividing by contact inhibition, it disappears once the cells are provided adequate growth space and start dividing again (139). Hence, while lipofuscin accumulation may indicate cell dysfunction and correlates with decreases in replicative potential, it seems unlikely that lipofuscin causes ageing since this accumulation is reversible and lipofuscin does not accumulate in all cell types. FREE-RADICAL THEORY

According to this theory, the production of highly reactive free radicals, such as the superoxide molecule, results in the progressive destruction of cytoplasmic and nuclear components and causes gene dysfunction resulting in senescence (140). Superoxide molecules are generated by reactions in the mitochondrial electron-transport system and may also be produced through the effects of UV and other forms of radiation, metal ions, and other factors. Lipids and proteins in cell membranes are particularly vulnerable to the action of free-radicals--and free-radicals can cause DNA damage. Catalase, peroxidase, and superoxide dismutase are among the scavenging enzymes which reduce the concentration of free-radicals. Some of the reactions which result in reduced levels of free-radicals are glutathione dependent. Tissues from old rodents appear to have low glutathione levels, low glutathione reductase activities, and reduced superoxide dismutase activities (141-144). Antioxidants, such as vitamin E, reduce free-radical levels and also protect against lipofuscin accumulation. Additional evidence for a link between freeradical production and ageing has been reported (145). DNA bases are oxidized by exposure to superoxide free-radicals. Oxidized thymidine residues are excised by DNA repair enzymes and thymidine glycol excretion reflects the amount of thymidine oxidized and the rate of DNA repair. The excretion of these products (as a function of animal weight) correlates with metabolic rate among many mammalian species. Free-radical production is also related to specific metabolic rate. Furthermore, excretion of oxidized bases in mammals is inversely related to maximum lifespan of the species. These results are 70

consistent with the hypothesis that free-radical damage to DNA has a role in the ageing process, but do not constitute proof of causality. It is possible, for example, t h a t some o t h e r m e c h a n i s m causes changes in the regulation of genes responsible for production of enzymes involved in the control of free-radical production or removal, and that changes in free-radical levels, if they occur in mammals, could be a consequence of senescence rather than its cause. MOLECULAR CLOCKS

Senescence could be caused by a developmental clock; perhaps the same clock controls the timing of events during maturation. The senescent phenotype could result from the chain of events initiated by the action of this clock as well as from the effects of lifelong exposure to disease and environmental influences. The developmental clock may trigger the production of cellular senescence factors. The developmental timing mechanism could involve clocks located in individual cells, could be a whole organism clock [analogous to the suprachiasmatic nuclei which control circadian rhythms in mammals (146,147)], or could include a hierarchy of clocks at the whole organism, tissue, and cellular levels which entrain one another (1). The operation of the clocks could be genetically programmed, could operate stochastically, or could function through a combination of random and programmed processes. The clocks may operate by counting cell divisions, as oscillating clocks, or as continuous clocks. Individual mammalian cells contain a clock which determines the maximum number of cell divisions they may achieve. Malignant transformation seems to interfere with this process, since fully transformed cells are "immortal" with respect to their replicative potential (27). Cellular immortalization by transformation can involve alterations in the function of only one or a few genes which usually are involved in the function of growth factors (148); thus the operation of the cell-division clock can be disrupted by quite simple alterations. A cell-division clock could be involved in the control not only of the cessation of cell division, but also in the timing of differentiation (42). O-2A progenitor cells differentiate into oligodendrocytes when cultured on a monolayer of type 1 astrocytes. The progenitor cells divide from one to eight times before differentiation occurs. Sister progenitor cells undergo an identical number of doublings before differentiating when they are subcultured. This implies the existence of a cell division counting clock which explains the timing of differentiation of oligodendrocytes and perhaps in other cell types as well. This type of clock could be important in development and senescence. Oscillating clocks have been described in Acetabularia, Euglena, and other organisms (149,150). The clock in Acetabularia and other green algae controls circadian rhythms, depends on periodic producCLINICAL BIOCHEMISTRY, VOLUME 25, APRIL 1992

BIOCHEMISTRYOF SENESCENCE tion of a 230-kd protein, and can be phase shifted by exposure to cycloheximide (150). The clock in Euglena and duckweed also controls circadian rhythms and operates through a series of reactions involving NADPH, calcium, and calmodulin (149). The reactions are dependent on one another and have negative and positive feedback effects which combine to result in a net reaction which oscillates with a 24-h period. These oscillating clocks control short-term cyclic functions, and would have to be associated with a "counter," or would have to be summated in such a way as to have an effect on long-term temporal functions. Continuous cellular clocks, if they exist, could operate through the accumulation or loss of factors which affect cellular function after these factors reach a critical concentration, or through progressive change in a cellular component which has an effect after a critical level of change has occurred. An example of the former process could be accumulation of D-aspartic acid (151,152). Metazoan cells produce only L-aspartic acid, but D-aspartic acid appears with a half-time of 85 years through a nonenzymatic, purely thermodynamic process of racemization. Thus, it is conceivable that D-aspartic acid could have a time-dependent effect on cellular function. An example of a progressive change in a cellular component which could result in time-dependent changes in cellular function is the nonenzymatic glycosylation of amino groups of DNA bases (74). Glycosylation of DNA bases causes changes in the physical characteristics of DNA, such as melting temperature, which are similar to DNA physical characteristics in late-passage fibroblasts and which would be expected to have effects on cellular function. Similarly, oxidative damage to DNA or other damaging processes could cause progressive changes in DNA resulting in changes in cellular function and senescence (145). However, such damage, if it occurs, must only affect regulatory regions of DNA and not structural DNA, since the primary structure of newly synthesized proteins in cells from old animals is the same as from young animals. Loss of telomeric DNA with successive cell divisions is also a potential mechanism through which a cellular clock could operate (93). Possibly, the event which initiates senescence is one or another form of cellular clock. This clock could affect the regulation of a family of genes, with effects on proliferation, differentiation, and other cellular functions. Changes in cell function so induced, together with disease, environmental, and other random influences, would combine to result in the senescent phenotype. Conclusions

The nature and causes of senescence remain elusive. Nevertheless, some promising leads to an understanding of senescence have been discovered over the past few years including the existence of cellular senescence factors, changes in DNA structure, inCLINICAL BIOCHEMISTRY,VOLUME 25, APRIL 1992

cluding alternations in DNA glycosylation and telomere length, and knowledge about processes which could be related to senescence, such as mechanisms of development in early life and operation of molecular clocks which control short-term rhythms. Many difficulties have beset progress in understanding the biochemical mechanisms of senescence. Often, exciting conclusions concerning senescence have been made based on studies using very new biochemical techniques in systems only just beginning to be understood. Once more is known about the limitations of the techniques being used or about the system being studied, some conclusions based on early observations have had to be modified or rescinded. For example, many felt that changes in DNA methylation patterns could cause senescence, but once more was understood about the role of DNA m e t h y l a t i o n p a t t e r n s , it became a p p a r e n t t h a t changes in these patterns with age were likely to be an epiphenomenon. The truth probably lies somewhere between these two extreme views, and one has to be careful to temper excitement about new findings using new techniques in recently discovered systems. On the other hand, one must avoid completely discrediting and forgetting about such findings if they have to be modified subsequently. Currently, m a n y investigators engaged in the study of senescence are concentrating on molecular genetic approaches which are yielding important new insights. The most important step will be to learn about the timing of developmental processes, which may only be understood by using complex experimental approaches. However, processes which are universal or widespread among species often turn out to be both simple and conserved, and it is possible that an uncomplicated process is responsible for initiating the causal chain of events (including changes in gene regulation) which lead to the manifestations of senescence. This fundamental process, which could be active and programmed, m a y turn out to be the same process which triggers earlier developmental events. Alternatively, senescence could prove to be caused by a random accumulation of events. There is considerable debate about whether senescence is programmed or random. The programmed nature of the process is evident from the heritability of maximum lifespan and the existence of cellular senescence factors. The process must, however, also have a stochastic nature since there is considerable interindividual variation in the timing and features of senescence. Several interventions appear to modify the timing of the initiating event or the subsequent steps which ultimately lead to phenotypic senescence. Interventions which slow biochemical processes, such as decreasing temperature (e.g., hibernation in nonobligate hibernators, freezing of cultured cells) and food restriction (reviewed in ref. 153) result in delayed manifestations of senescence. Cellular transformation can circumvent senescence altogether and meiosis starts the process of ageing over again. Hence, even now, the rate of senescence can be modified. 71

KIRKLAND E v e n t u a l l y , not only m a y t h e i n i t i a t i n g cause of senescence be discovered, b u t it m a y become possible to control t h e r a t e of senescence in complex organisms.

Acknowledgements The author wishes to thank P. Smith for her assistance in the preparationof this manuscript. This work was supported by Medical Research Council of Canada Grant M A 7679 and the Queen Elizabeth Hospital Foundation.

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