Chromatin reorganization during senescence of proliferating cells

Mutation Research, 256 (1991) 81-104

81

© 1991 Elsevier Science Publishers B.V. All rights reserved 0921-8734/91/$03.50

MUTAG1 00153

Chromatin reorganization during senescence of proliferating cells A. Macieira-Coelho Laboratory of lmmunology, Medical Faculty (Piti~-Salp~triOre), 75013 Paris (France)

(Accepted 3 June 1991)

Keywords: Chromatin; Genes; Genome organization; Ageing; DNA anchorage

Summary It was previously proposed (Macieira-Coelho, 1979) that aging of proliferating cells is the result of genome reorganization taking place during the division cycle. This hypothesis was investigated and a reorganization could indeed be ascertained in the different hierarchical orders of D N A structure; a correlation was found between changes in chromatin organization and the impairment of cell cycle-related events. Indeed, like the latter, the reorganization of chromatin structure is characterized by a succession of subtle changes through the cell population life span, and a final short stage with abrupt events. The final events seem to concern mainly the organization of heterochromatin. The reorganization in the genome is accompanied by structural changes in the cellular scaffold and an evolution of cell morphology. The remodeling occurring in the cell through serial divisions seems to take place in such a way as to decrease the probability of further reorganizations, tending to a limit. The decline of the proliferative activity seems to be the result of the tendency to reach this limit.

The organization of DNA in the nucleus In order to understand the implications of chromatin reorganization for the deregulations occurring during cellular senescence, a reminder of what can be called the mechanics of chromatin function is necessary. Mammalian D N A is a molecule more than 1 m long, confined in a sphere, the nucleus, with a diameter of about 5 /~m. This implies an elaborate folding which is regulated inter alia by the

Correspondence: Dr. A. Macieira-Coelho, INSERM, 73 bis rue Mar6chal Foch, 78000 Versailles (France).

structure of D N A (Crothers et al., 1990), by D N A bound proteins, by enzymes and by the anchorage of this make-up to a protein matrix. In addition there are glycoproteins present (Zardi et al., 1976) whose role is yet to be determined; since these molecules are highly charged, it is reasonable to think that they must also function as regulators of chromatin conformation. Considerable knowledge has been gathered with regard to the biochemical reactions affecting the binding of histones to DNA, which is responsible for their action on the folding of the double helix. Several biochemical events are involved in the regulation of DNA-histone binding, one of them

82 is the covalent attachment of branched chains of ADP-ribose to nuclear proteins (ADP-ribosylation). It weakens the binding (mainly of histone H1) through an increase in the protein negative charge and probably also by distorting the chromatin structure through the presence of the long branched chain (Kanungo, 1980). Acetylation and methylation are two other biochemical reactions regulating protein-DNA binding; the former, decreasing the net positive charge, would also dissociate the binding whereas methylation would render the binding stronger through an increase of the positive charge. These two reactions seem to act mainly on nucleosomal core histones. A fourth biochemical reaction controlling protein-DNA binding is phosphorylation which is the general mechanism used by the cell to vary the configuration of molecules and thus regulate their potential to intervene in metabolism and their response to different effector molecules. Alterations in molecular shape are the basis of all cellular life processes. The loss of molecular and cellular conformational flexibility seems to be an important parameter of senescence. Less is known about the role of the non-histone proteins in the preservation of the flexibility of the high order structure of DNA; it seems, though, that they participate in the assembly of nucleosomes and stabilize histone octamers (Stein et al., 1979). In general, at least three hierarchical levels of nuclear D N A organization resulting from DNA folding are accepted (Comings, 1978), although more have been postulated (Nicolini, 1983). The first level corresponds to the 10-nm 'bead-on-astring' chromatin fiber that results from the repeating unit formed by two superhelical turns of the DNA double helix around a histone protein core. The unit is called the nucleosome and the number of base pairs of the repeating structure is called the DNA repeat length. The second level results from the folding of the 10-nm fiber into a 30 nm wide solenoid with a helical pitch of 11 nm and 6 - 8 nucleosomes per turn (Finch and Klug, 1976). There is no complete agreement concerning the organization at this level (Walker and Sikorska, 1987). Finally the third level results from the further folding of the solenoid into supercoiled loops, each with ap-

proximately 10,000 bp of DNA, anchored at the periphery of the nucleus (Pardoll et al., 1980). This elaborate organization of the DNA molecule is fundamental for gene expression; it is thermodynamically unstable and thus is in permanent movement and variance, the structural flexibility being crucial for its function. The difficulty in identifying the exact nature of the reorganizations that can take place in this complex arrangement is hardly surprising. An additional complication comes from the possible differences in the higher order packing of the nucleosomal chain depending on the cell type (Zentgraf and Franke, 1984). This is just a brief description of the organization of nuclear DNA; a more detailed one would not be within the scope of this review. Those interested in a thorough review of the state of the art concerning chromatin can find it in a recent publication (VanHolde, 1989). As mentioned above, the scaffold upon which chromatin is anchored plays a crucial role in its high order structure. There is indeed a protein framework called the nuclear protein matrix with which DNA has been found to be associated (Berezney and Coffey, 1975). Pertinent to the subject that will be discussed below is the finding that the DNA synthesis initiating sites are preferentially located at the borders between condensed chromatin and interchromatin areas (Berezney and Coffey, 1975), suggesting an important role of the nuclear matrix, in particular the peripheral nuclear region, in the initiation of the replication of DNA. The anchorage of DNA is crucial not only for replication but also for transcription, since nascent RNA is associated with the nuclear cage (Jackson et al., 1984). The nuclear lamina, a filamentous protein meshwork lining the nucleoplasmic surface of the nuclear envelope, probably provides an anchoring site at the nuclear periphery for interphase chromatin (Gerace, 1985). When the nuclear shell is isolated, it contains chromatin structures made of packed nucleosomes 28-32 nm thick that are associated with the three nuclear lamins (Bouvier et al., 1985). The lamina is composed of proteins called lamins which seem intermediary structures between DNA-binding proteins and the cytoskele-

83 ton. Indeed on the one hand the lamina is tightly bound to chromatin since it can be dissociated from chromatin only by high salt solution which also extracts the tightly bound histones in the nucleosome cores (Bouvier et al., 1985). On the other hand, lamins have a striking sequence homology with intermediate filaments, a component of the cytoskeleton (Gerace, 1985). Evidence in favor of the influence of intermediate filaments on chromatin conformation was recently reported (Hay and Deboni, 1991); it was shown that the disruption of intermediate filaments induces chromatin motion in neuronal interphase nuclei. So the anchorage of chromatin seems to be fulfilled with t h e preservation of the continuity with the cytoplasmic scaffold. This way D N A is linked to the cytoskeleton through its anchorage to the nuclear cage and via the former to the cell membrane and the extracellular matrix. This whole structure has to be seen as a tridimensional manifold where the information flows to a great extent through topological constraints. As a matter of fact experimental evidence favors this view (Macieira-Coelho, 1983). Plating cells on surfaces whose physico-chemical properties could be modified in a controlled fashion allowed the modulation of cell morphology, proliferation, differentiation and malignancy (Macie i r a - C o e l h o and A v r a m e a s , 1972, 1973; Macieira-Coelho et al., 1974; Wahrmann et al., 1981). The modulation of the cell phenotype by cell-substratum interactions seemed to be mediated through changes in gene expression, the first signal transmitted from the periphery being of a physical nature (Macieira-Coelho et al., 1974; Macieira-Coelho, 1988a). These works and others relating the cytoskeleton with the initiation of D N A synthesis (see for review Macieira-Coelho, 1983) led to the conclusion that cell movements originating at the cell membrane are propagated through the cytoskeleton to the nucleus where they contribute for chromatin to assume a favorable conformation for the initiation of DNA synthesis. This mechanism is impaired during cell aging because of the alteration of the cellular scaffold. As described below, there is a relationship between the age-related reorganization of chro-

matin and the disturbance of this information flowing between the cell membrane and the genome. Rationale for the search for chromatin reorganization in proliferating cells The observation that there is a direct relationship between the frequency of chromosomal recombinational events and the long-term proliferative potential of fibroblast cell populations produced a new paradigm (Macieira-Coelho, 1979, 1980, 1988b, 1990). It was postulated that the progressive shift in cell behavior leading to senescence during serial proliferation is due to the genome reorganization which inevitably accompanies cell division and modifies D N A at different levels of its structure. This could be due inter alia to chromosomal rearrangements, deletions, sister-chromatid exchanges, illegitimate recombination, DNA loss, displacement of transposable elements, gene amplification and other phenomena that occur in the genome during the division cycle. It depends on intrinsic properties of the genome involved and can be accelerated by external events (physical, chemical or biological). This reorganization, although providing the cell with a way to evolve, adapt and survive, would also determine its permanent drift and would occur in such a way as to decrease the probability for further reorganizations, tending to a limit. Aging of dividing cells would be the result of the propensity to reach that limit. There is a considerable amount of data showing that at the level of chromosomes, a reorganization of the genome takes place during serial cell divisions. Chromosomal translocations, inversions, dicentrics, breaks, deletions and chromosomal losses have been shown to increase during aging (Jacobs et al., 1961; Saksela and Moorhead, 1963; Benn, 1976; Miller et al., 1977; Pierre and Hoagland, 1972; Galloway and Buckton, 1978). Ploidy has also been reported to evolve, with a decline of 2C DNA content and an increase of 4C nuclei (Matsuo et al., 1982). Furthermore, chromosomal structural aberrations have been found to increase significantly in older individuals (Hedner et al., 1982; Martin and Rademaker, 1987). Prieur et al. (1988) were able

84 to distinguish between chromosomal rearrangements due to development and those related to aging. The latter were attributed to the exposure to mutagens. Indeed it is not only the spontaneous fragility of chromosomes that increases with aging; chromosomes in lymphocytes from old donors are also more susceptible to induced damage (Dutkowski et el., 1985; Kishi et al., 1987; Esposito et el., 1989). A comparative analysis of cytogenetic studies performed on different cell types suggests that the long-term doubling potential of cells is directly related to the potential for continuous chromosome rearrangements, in other words, with the plasticity of the genome. Indeed human embryonic fibroblasts, when cultivated in vitro, go through continuous chromosomal rearrangements without any definite pattern becoming predominant (Chen and Ruddle, 1974; Harnden et el., 1976). These cells have a longer division potential than postnatal fibroblasts which go through more stable clonal-type chromosomal rearrangements during serial divisions (Harnden et el., 1976). In other words, both the plasticity of the genome and the division potential decrease during development. On the other hand, fibroblasts from Werner's syndrome patients, which have a reduced division potential compared to cells from age-matched normal donors, present chromosomal rearrangements which become predominant and fixed during serial proliferation (Salk et el., 1981); this pattern of chromosomal rearrangements characteristic of Werner's syndrome was called variegated translocation mosaicism. These findings give further support for a correlation between the long-term potential for proliferation and the 'plasticity' of the genome, which declines during development and aging and is diminished in Werner's syndrome. The study of the effect of ionizing radiation on different types of cells gave further support to the relationship between cell survival and the recombination potential of the genome. It was found on the one hand that radiation induced recombinational events; most of the breaks involved in exchanges (53 out of 62) concerned the centromeric and telomeric regions (Bourgeois et el., 1981). Thus the intrachromosomal break distribu-

tions where radiation-induced exchanges took place were preferentially located at regions rich in repetitive DNA, which has been implicated in recombinational events (Schmid and Jelinek, 1982). On the other hand, radiation could prolong, shorten or have no effect on the proliferative potential and this response was found to be directly related to the potential for genetic recombinations of the cell populations involved (Bourgeois et al, 1981; Macieira-Coelho, 1990). Furthermore, the propensity of fibroblasts of different origins to overcome the growth decline and to immortalize seems to be directly related to the potential for chromosomal recombinational events (Macieira-Coelho, 1980, 1990). The rearrangements occurring during the division cycle at the level of the chromosomes must be only the visible part of the iceberg and one may wonder what happens at other levels of DNA organization (Macieira-Coelho, 1984). Indeed other cytogenetic studies are suggestive of structural modifications in chromatin, during the chromosomal rearrangements that accompany aging. Thus, loss of centromeres could be ascertained in the lymphocytes of aged women, which could be one of the causes of the increased nondisjunction (Nakagome et al., 1984). Another interesting cytogenetic finding was the decline of silver-stained nucleolar organizer regions which seems to be determined by heterochromatization of satellite stalks (Lezhava, 1984). These regions contain genes coding for 18S and 28S ribosomal RNA and hence the results could be germane to the reported loss of genes coding for ribosomal RNA in aging cells (Johnson and Strehler, 1972). Finally, the discovery that the partition of DNA at the end of the division cycle is asymmetric in a significant fraction of cells (Macieira-Coelho et el., 1982) also suggested that proliferation is a source of genomic reorganization and hence of a genetic drift relevant to the understanding of the mechanisms of aging. These studies showed that DNA synthesis is not semiconservative when analyzed at the level of the individual cell and further supported the rationale to search for genomic reorganizations at the molecular level. The asymmetry in DNA distribution at the time of cell division could explain in part the increased variance with age in the DNA content of human

85

increasing PDL, DNA becomes more thermolabile since it sediments with a lower molecular weight, revealed by a shift of the peak to the left when cell lysis was done at 37 ° C. At the very end of the cell population proliferative potential, D N A of the heated preparations sediments in a dispersed fashion. These results suggest structural modifications rendering chromatin progressively more fragile. In the virtual postmitotic cells, low molecular weight DNA is present in the cells lysed at room temperature, since it sediments with two peaks, one with a molecular weight identical to the peak of younger cells and the other with a molecular weight identical to A phage (circular) DNA (Puvion-Dutilleul et al., 1984). Additional evidence favoring an increased fragility of the chromatin structure during serial proliferation of human cells was obtained by centrifuging chromosomal D N A from human fibroblasts at different PDL, in alkaline sucrose gradients after lysing at room temperature with sarcosyl and sodium deoxycolate (Fig. 3). DNA sedimented in an increasingly dispersed fashion directly related to the PDL, showing an increase in sensitivity to strong detergents, which suggests

blood mononuclear cells (Staiano-Coico et al., 1982).

Structural changes in chromatin during aging of proliferative cell compartments A progressive increase in alkaline labile sites during serial proliferation of human embryonic fibroblasts, detected by sucrose gradient centrifugation, was reported by Icard et al. (1979). These results suggested the presence of D N A singlestrand breaks; double-strand breaks could not be detected with sucrose gradient centrifugation at neutral pH. Subsequently it was found that the methodology used heated the D N A preparation and that the breaks present in cells before the terminal stage (phase IV) were due to an increased chromatin thermolability (Puvion-Dutilleul et al., 1984). Fig. 1 illustrates the survival curve of the cell population used and Fig. 2 the effect of temperature on the profile of chromosomal D N A from cells at the 20th, 52nd and 60th population doubling levels (PDL), centrifuged in alkaline sucrose gradients after lysing the cells during 18 h at 20 and 37 °C in sodium dodecyl sulfate. With II

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Fig. 1. Maximal cell densities recorded before each subcultivation during the entire life span of a h u m a n embryonic lung fibroblast line. Each dot corresponds to a cell count from a different culture vessel. Phase I concerns the first 5 doublings when the cell densities increase due to selection of the fibroblast population and elimination of other cell types. Phase II lasts from the 5th to approximately the 43rd doubling when close to 100% of the cells enter D N A synthesis during a 24-h period and the population doubling time is stable. Phase III lasts from the 43rd to around the 55th doubling when still close to 100% of the cells synthesize D N A during a 24-h period but the population doubling time increases. Phase IV corresponds to the last 3 - 5 doublings when the n u m b e r of cells capable of synthesizing D N A during a 24-h period declines rapidly and the doubling time increases pronouncedly.

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modifications of p r o t e i n - D N A binding. Further evidence for the latter defect was obtained by following the sedimentation velocity of newly syn3000 2000 1500 1000 800

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thesized D N A after heating the cells during lysis (Icard-Liepkalns and Macieira-Coelho, 1982). Fig. 4 illustrates the sedimentation velocity of D N A at different times after stimulation and addtion of 3H-thymidine to cultures of young and old cells. In both cultures, 45 min after labeling the radioactivity is distributed in small peaks through the gradient. Then D N A progressively sediments in young cells as a single peak which is completed 10 h after labeling, i.e., after covering the time of a S period. In old cells, however, even at the 16th h after addition of 3H-TdR, D N A sediments in several peaks with different sedimentation velocities although most of the cells had gone through the S period (Icard-Liepkalns and MacieiraCoelho, 1982). These results could be due to a defect in the gap-filling step during the rejoining of adjacent replicons during the G 2 period (Van't Hof, 1980) because of the disturbances in the metabolism of chromosomal proteins found in old cells (Ryan and Cristofalo, 1972; Srivastava, 1973; Stein, 1975; Pochron et al., 1978; Mitsui et al., 1980). The data could explain the prolongation of the G2 period that characterizes senescence of proliferative ceils (Macieira-Coelho and Taboury, 1982) and constitute an example of the functional

87

implications of the disturbances in chromatin organization during the division cycle. The relationship of this defect to changes in protein-DNA binding is supported by experiments showing that hydrocortisone, which can

retard the decline of the proliferative potential of these cells, can also correct the disturbance of the gap-filling step. The results illustrated in Fig. 5 show that D N A sediments in a single peak much earlier in the cells treated with hydrocortisone.

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Fig. 4. Radioactivity collected from the different fractions after alkaline sucrose gradient centrifugation of D N A from resting cells taken at the indicated times after stimulation. The dashed line indicates the fraction where denaturated A phage D N A (40.5S) sedimented. Young cells were at the 27th PDL and old cells at the 52nd PDL and are identical to those used in the experiments illustrated in Figs. 2 and 3. The sedimentation direction was from left to right.

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Fig. 5. Experiment identical to that illustrated in Fig. 4 but performed with ceils at the 37th PDL without and with hydrocortisone during the 10 preceding population doublings. Steroid hormones are indeed known to stimulate the synthesis of D N A - b o u n d proteins and the acetylation and phosphorylation of histones and nonhistone chromosomal proteins (Kanungo, 1980) which are important for the stabilization of the links between adjacent replicons. Mayer et al. (1986) could not find any evidence favoring the presence of D N A breaks in cells before the terminal stage. More recently, however, Dayton et al. (1989) reported that senescent fibroblasts have an increased propensity for single-stranded D N A breaks. The evidence obtained was of an indirect nature, through the detection of single-strand breaks in a transfected plasmid. It was postulated that they were due to an increased nuclease activity that may be responsible for the D N A damage observed in senescent cells.

The presence of low molecular weight D N A could be ascertained during aging of another proliferative compartment, i.e., human peripheral blood cells (Turner et al., 1981). Differential scanning calorimetry ascertained a decline of the melting temperature of human fibroblast DNA, after serial proliferation, suggesting an increase in single-stranded D N A followed by strand breaks that destroy its supercoiling potential (Almagor and Cole, 1989). After immortalization the cells have melting profiles identical to those of young cells. Mouse fibroblasts evolve in a way identical to that of human cells, since D N A sediments with a dispersed pattern after alkaline sucrose gradient centrifugation when lysis is done at room temperature, only from ceils in growth crisis (Figs. 6 and

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Fig. 6. Survival curve of a newborn mouse lung fibroblast population expressed as the maximal cell densities (• . . . . . . •) reached before each subculture during the growth decline, growth crisis and recovery. The bars indicate the fraction of slow, m e d i u m and rapid dividers, i.e., the cells that performed one ( I ) , two (t~) and three (~q) divisions respectively, in the presence of 5-bromo-24-deoxyuridine. W h e n the cells immortalized, the population consisted only of rapid dividers.

weight DNA had disappeared, the genome had gone through profound reorganizations since the karyotype had switched from diploidy to tetraploidy (Fig. 8).

7). These breaks, however, disappear when the cells immortalize which could be due to the elimination of the damaged DNA (Macieira-Coelho and Azzarone, 1989). At the time low molecular

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Fig. 8. Chromosome number found at different PDL in cells identical to those used in the experiments illustrated in Figs. 6 and 7.

On the other hand, serially proliferating adrenocortical cells do not seem to accumulate DNA damage (Hornsby and Harris, 1987). The ultrastructural analysis of chromatin from human fibroblasts also suggested reorganizations developing during serial proliferation. Cells in phases I, II and III fixed with glutaraldehyde alone (Brock and Hay, 1971; Puvion-Dutilleul and Macieira-Coelho, 1982) showed the chromatin condensed at the nuclear periphery and around the nucleolus and largely dispersed in the nucleoplasm (Fig. 9). In terminal cells (phase IV), the nucleoplasm appeared clearer and the nucleolus was enlarged. Staining with uranyl and lead and DNA specific staining (Fig. 9B) showed that chromatin from phase IV cells was accumulated neither along the nuclear envelope nor around the nucleolus and displayed a higly dispersed pattern. Since the chromatin condensing at the nuclear periphery and around the nucleolus is supposed to correspond to heterochromatin, it is possible that the latter is affected mainly in the terminal (differentiated?) cell. Since removal of H1 is known to lead to chromatin decondensation (Oudet et al., 1975), the changes observed in the terminal cell under electron microscopy could be related to the alterations in the synthesis of H1

histone detected in postmitotic cells (Mitsui et al., 1980). A change in the synthesis of the H2A histone variant has also been reported (Dell'Orco and Worthington, 1988). The 30-nm solenoid was visualized with a loosening procedure (Puvion-Dutilleul and MacieiraCoelho, 1982) which revealed striking modifications in the organization of the fibers in the terminal cell (Fig. 10). The nuclei were abnormally clear, the chromatin threads were rarer than in the young cells and at the nuclear peripho ery the threads were shorter and unusually spaced along the lamina densa which sometimes was entirely devoid of chromatin threads. The nucleolar filamentous masses displayed a granular appearance due to the knobby configuration of their entangled filaments which had lost the fine network organization characteristic of the young cells. The fibrils which constituted the nucleolar masses of young nuclei were 6-7 nm thick whereas they reached 10-15 nm in old nuclei. The frequency of nuclei with altered chromatin fibers, from cells at different PDL, was determined by direct screening of electron microscope preparations from cells identical to those used for Fig. 10 (Table 1). It was found that up to the 41st PDL, altered nuclei could not be detected; from the 41st to the 49th doubling, 5% of the nuclei had a modified organization of chromatin and from the 50th doubling onward to the end of the cell population proliferative lifespan the proportion of altered nuclei increased abruptly from 5 to 98% (Puvion-Dutilleul et al., 1982). This shows that the rapid fall in the fraction of cells synthesizing DNA per unit of time is accompanied by dramatic changes in the nucleus simultaneous with the increase in cell size. Identical measurements were made in early passage skin cells obtained from two human embryos, from three normal donors of different ages (MVL, AG4353, AG4059) and from one 60-yearold patient with Werner's syndrome (AG780) (Table 2). Nuclei with gross changes, visible by direct observation, were absent in fetal cells but could be seen in normal postnatal fibroblasts from the 30- and 59-year-old donors; they were increased in cells from a 96-year-old donor as compared to middle-aged donors and in cells from the patient as compared to the age-matched

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Fig. 9. Nucleoprotein organization following conventional fixation with glutaraldehyde alone of phase II (A) and phase IV (B) cells identical to those whose survival curve is illustrated in Fig. 1. × 18,000; bar = 1 ~zm. (a) Uranyl and lead staining; chromatin is condensed at the nuclear periphery (arrows) in phase II cells and is more dispersed in the nucleoplasm of phase IV ceils, which appear clearer than in young cells. The nucleolus (Nu) is prominent and presents a bud (arrow). (b) Specific D N A staining; dense chromatin is accumulated at the nuclear (arrows) and nucleolar (arrow heads) peripheries of young cells but not of old ones. In addition, the reaction shows chromatin to be less dispersed in the nucleoplasm of younger than of phase IV cells. (c) Preferential R N P staining; in young cells the bleached chromatin is mainly located at the nuclear periphery and around the nucleolus (arrows). The latter (Nu) and the nucleoplasm which contain R N P structures remain heavily contrasted. In phase IV cells the nucleolus is well contrasted whereas non-nucleolar R N P structures are more dispersed than in early P D L cultures, consequently the nucleoplasm appears clear.

92

Fig. 9B (continued).

control (Puvion-Dutilleul and MacieiraoCoelho, 1983). In addition, the cells from the 96-year-old normal donor and from the 60-year-old patient had altered nucleoli. Thus it seems that ceils with old-type chromatin appear during development and that a pronounced increase of these cells

occurs only late during the human life span and earlier with pathological aging. The same can be said of the nucteolar alterations. Pictures like those illustrated in Fig. 10c and d, of the periphery of nuclear preparations from cells at different PDL, were screened with an

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Fig. 10. 30-nm chromatin fibers following mild loosening of phase II (a and c) and phase IV (b and d) cells identical to those used for Fig. 1. Nucleolar (a and b) and peripheral nuclear (c and d) regions.

94 TABLE 1 PERCENT NUCLEI WITH ALTERED NUCLEOLI AND CHROMATIN FOUND AT DIFFERENT PDL DURING THE SERIAL SUBC.ULTIVATION OF THE HUMAN EMBRYONIC LUNG FIBROBLAST LINE ICIG-7 PDL

%

10-40 41-49 50-56

0 5 98

0.4

'

"~

~z

'

~

i

~I

0.1

LU

¢~

image processor (Macieira-Coelho and PuvionDutilleul, 1989). Two measurements were made, one expressed the ratio between the dark and light areas and was called the density of the fibers; the other, which was called the spacing, was obtained with a sieve-like procedure that calculated the areas between the fibers. The density of the fibers, mainly at the level of their anchorage to the lamina densa, was found to decrease progressively (Fig. 11). On the other hand, the spacing of the fibers was found to follow a two-step pattern; it varied very little during most of the cell population life span and increased abruptly at the end when the cells entered the terminal postmitotic stage. These modifications in the organization of the 30-nm solenoid fibers have functional implications. Indeed the fall in the density correlated with the decline in the rate of D N A synthesis initiation after cell attachment and spreading (Fig. 12), whereas the evolution of the spacing correlated with the terminal fall in the maximal number of cells capable of initiating D N A synthesis during a 24-h period, under conditons where the

80 706050~: 40~ 30~" ~0|0i

~ 1~ 2~ 3'2 4~) 4'$ ~'6 ' POPULATION DOUBLING Fig. 11. Plot of the values for the density and spacing of the 30-nm chromatin fibers obtained with an image analyzer, found in human fibroblasts at different PDL identical to those used for Fig. 1. The bars represent the 95% confidence limits; the vertical dashed lines indicate the approximate limits of phases II, III and IV.

cells have built their attachment sites and are freely migrating on the substratum. Hence density and spacing must correspond to two different

TABLE 2 PERCENT NUCLEI WITH ALTERED NUCLEOLI AND CHROMATIN FOUND IN LOW PDL CULTURES OF HUMAN SKIN FIBROBLASTS Cell line Fetal

ICIG-9 AG4525

Postnatal

MVL AG4353 AG4059 AG780 (Werner's syndrome)

Age (years)

PDL/Maximal number of doublings

Nucleoli

Chromatin

63 89

17 7 63 89

4 / ~ 50 6 / ~ 50 30 59 96 60

12/30 6/40 5/29 6/19

95 ~ I00-

x

~

,,

x 0.4 ¸ I

~

0.3 ¸

z ~

0.2.

500.1.

90-

o ne ~1 n

~ 8

~ 16

24

32

~ 40

48

70

POPULATION DOUBLINGS

~

60

~ ,< "u}

40

_z

Fig. 12. Percent labeled interphases found during the first 24 h after subcultivation when the cells have to build their attachment sites (©) and maximal percentage reached during a 24-h period thereafter ( × ) before the cells reach resting phase. The experiments were performed at different PDL on cells identical to those used for Fig. 1.

parameters within the high order structure of chromatin, the pronounced changes in the spacing corresponding to more profound reorganizations of chromatin structure, maybe associated with the decondensation of heterochromatin revealed after glutaraldehyde fixation, since both are terminal events. It is pertinent to remember that at high PDL there is a change in the timing of DNA replication in the centromere of chromosome 9, a region rich in heterochromatin (Lindgren and Farber, 1982). The measurements performed with the image processor were also made on fibroblasts cultivated from skin biopsies of normal donors of different ages and of two patients with Werner's syndrome (Fig. 13) (Macieira-Coelho and PuvionDutilleul, 1989). It was found that the density decreased in an inverse relationship with the age of the donor and that the values found for the cells of the Werner patients did not differ from the age-matched controls. The spacing increased in a direct relationship with the age of the donor and the values found for the cells from the Werner patients were significantly higher than for those of the age-matched control donors. This again suggests that density and spacing express different parameters of the high order organization of chromatin, only the spacing being disturbed in these cases of pathological aging. Additional evidence in favor of a reorganization at another level of DNA structure came from

1

80

| 56

50

30 20

10 10

50

lOOyears

Fig. 13. Plot of the values of the density and spacing of the 30-nm chromatin fibers obtained with an image analyzer, found in human skin fibroblast cells before the 4th PDL, from nine normal donors (~) and from 2 patients with Werner's syndrome (©) of different ages. The bars represent the 95% confidence limits.

studies performed on the 10-nm chromatin fiber. Micrococcus nuclease digestion of chromatin from postnatal human skin fibroblasts at different PDL (Dell'Orco et al., 1986) showed that the initial velocity of the reaction and the final plateau were unchanged; at intermediate times, however, the velocity was higher in older ceils (Fig. 14). These results are compatible either with an increased 2~C 205 20C ~ 19~ ._

g_~c ~ ~a"~

g~

18C ~7~ .

.

.

.

.

.

.

.

~7C 16~

3

2

4

6 8 IO Incubotion Time (Minutes)

12

14

16

Fig. 14. Digestion velocity with micrococal nuclease of DNA from low PDL (six experiments 13-23 PDL) (e e) and from high PDL (seven experiments 51-70 PDL) ( × - - - x ) postnatal human skin fibroblasts. Each value corresponds to the mean and the SEMs are indicated. (Reprinted with permission from Dell'Orco et al., 1986.)

96 170

reported (Ishimi et al., 1987). In addition, in spreads of Werner's syndrome nuclei, short pieces of unbeaded DNA fibers with lengths distributed between 0.1 and 0.2 /xm were frequently seen, occasionally forming a circle (Fig. 16c).

165

~ ~6o 5 o- ~55 ~3 150

I T

t45

2 a

i ~

,b

,'~

~ t

14C 155

~

,~

~

~

,'~

,t

Incubotion Time (Minutes)

Fig. 15. Size of the monosome fragment from low (o) and high ( × ) PDL cells determined from the same experiments illustrated in Fig. 14. (Reprinted with permission from Dell'Orco et al., 1986.)

heterogeneity of DNA repeat lengths or with conformational changes of the substrate. The possibility that the increased digestion velocity was due to nucleosome sliding in older cells during the incubation was ruled out by running the reaction at 4 ° C to inhibit the exonuclease activity of micrococcal nuclease and fixing the nuclei prior to digestion with formaldehyde to induce reversible DNA-protein crosslinks (Dell' Orco et al., 1986). When the size of the monosome fragment was analyzed for all digestion times with the cells of different PDL, the size of the fragment declined with longer digestion times to a constant value of about 146 bp after 7 min of digestion, identical for low and high PDL cells (Fig. 15) (Dell'Orco et al. 1986). These results indicate that the structural changes detected with the micrococcal nuclease (Fig. 14) are located to the linker region. The 10-nm fiber was also studied with electron microscopy (Puvion-Dutilleul and MacieiraCoelho, 1983). The fibers from fetal cells in phases I, II and III (Fig. 16a) and from young adult donors (not shown) displayed a typical nucleosomal organization whereas those from terminal fetal cells (not shown) and from older donors (Fig. 16b) were punctuated by widely spaced nucleosomes or were entirely extended. The changes observed with Miller spreads could be due to shear forces on a more fragile structure, resulting from the ionic detergent, the alkaline pH and the hypotonic solution used during the preparation for electr~" microscopy. A confirmation of these structural changes in the 10-nm fiber was later

Fig. 16. Miller spreads performed with human skin fibroblasts of embryonic origin (a), from a normal 59-year-old donor (b) and a 60-year-old patient with Werner's syndrome (c). Bar = 0.5/xm.

97

These changes, like those mentioned above, may also be secondary to alterations of the synthesis of histone H1 (Mitsui et al., 1980) which plays a crucial role in stabilizing the 10 additional bp at both ends of the 140-bp core particle (Simpson, 1978). Other modifications of DNA-bound proteins concern the accumulation of residual acidic proteins (Mitsui et al., 1980), the synthesis of two new major proteins that are not present in young cells and the loss of the ability to synthesize detectable amounts of four major nuclear proteins that are found in young cells (Sakagami et al., 1979).

A

It was reported that the positioning of nucleosomes may be important to maintain the condensed nature of heterochromatin (Igo-Kemenes, 1985); it would create a spatial arrangement of DNA favorable for chromosome positioning. Hence our findings suggesting a change in nucleosomal positioning could be related to the decondensation of heterochromatin found in the postmitotic cells (Fig. 9), and explain to a certain extent the sudden difficulty in the progression through the division cycle. Low molecular weight D N A was extracted from human fibroblasts at different PDL, separated on

B

C

Fig. 17. Electron micrograph of circular D N A from h u m a n embryonic fibroblasts at doubling 59, identical to those used for Fig. 1. D N A circles with 4.91 _+ 1.1 kb (A), 1.5_+0.4 kb (B) and 17.2_+2.4 kb (C); the latter is mitochondrial DNA. Length m e a s u r e m e n t s were made relative to the plasmid pBR322 (4361 bp). Magnification × 51,000.

sium chloride gradients and prepared for visuzation under electron microscopy (Icard-LiepIns et al., 1986). It was found that terminal, tual postmitotic cells displayed circular extraromosomal D N A mainly with 5 and 1.5 kb (Fig. ). These results fit the ones obtained with su9se gradient centrifugation showing the presce of low molecular weight DNA in postmitotic lls, which sediments with A phage DNA (Fig. The circles were found to contain sequences the interspersed highly repetitive KanI and ~ families (Riabowol et al., 1985; Icard-LiepIns et al., 1986) and sequences homologous to ~ a-globin and /3-actin genes (Icard-Liepkalns al., 1986). This shows that the circles are terogeneous and originate from different parts the genome. It is believed that during the division cycle, cular D N A detaches and reintegrates again in e genome. It is possible that in the postmitotic lls the circles do not reintegrate into the chro~somes because of the profound chromatin uctural reorganizations taking place. It is interIing that in plants, extrachromosomal D N A 91icates in cells that differentiate from G 2. It s been suggested that this is due to the failure nascent replicons to join when cells reach G 2, ~ving gaps that serve as recognition sites for the tiation of DNA amplification (Van't Hof and erknes, 1982). Hence, the defect described ove concerning the lability of the gap-filling :p during the G 2 period of phase IV (differenti~d?) cells could be an explanation for the aparance of extrachromosomal circular DNA. Amplification of extrachromosomal circular ~IA was also found to increase in human lymocytes during aging of the organism; the ampliation of restricted size classes of circular DNAs ems to occur only in the later part of the man life span (Kunisada et al., 1985). Circular qA was also found to be present in the lympho:es of Werner syndrome patients although not lplified when compared with cells from age~tched normal donors. In a murine model of :elerated senescence, it was found to be incased (Kunisada et al., 1985). Polydisperse circular DNAs are present in ndividing cells; their possible relationship with velopment and aging has been investigated

(Flores et al., 1988). It was found that small polydisperse circular DNAs showed similar size distributions at all ages in mouse heart tissue; however, more discrete size classes and slightly larger circles were observed in the tissue from older mice. An age-related inhibition of DNA digestion by DNase I in nuclei from older fibroblasts is also suggestive of chromatin conformational changes developing during serial proliferation (Dell'Orco and Whittle, 1982). Conformational modifications are also apparently due to an increase in disulfide bonds (Tas et al., 1980), another element that helps sustain chromatin structure. Age-related conformational changes of DNA were also reported in human peripheral blood lymphocytes (Hartwig and K6rner, 1987). A decreased transcription accompanies the chromatin reorganization described above (Hill et al., 1978; Puvion-Dutilleul and Macieira-Coelho, 1982) (Fig. 18).

Relationship between chromatin reorganization and the changes of the supporting structures No studies have been made so far on the nuclear matrix during cell aging, but indirect evidence suggests that changes must indeed occur. Thus, in serial proliferating fibroblasts, the nuclear and nucleolar dry masses and areas increase (Bemiller and Miller, 1979). The patterns of increase are similar for all measures; the increases in nuclear and nucleolar areas are interdependent until the last doubling where they increase independently. The increase in nuclear area occurs in parallel with the decline of the density of the chromatin fibers and the independent increase in the two values coincides with the increase of the spacing, suggesting a coupling between these events. Furthermore, the increase in cell volume was found to be coupled, during aging of chicken fibroblasts, with the increased hindrance of the initiation of D N A synthesis (Lima and Macieira-Coelho, 1972). Fig. 19 illustrates the profound morphological modifications occurring through serial divisions of human fibroblasts that are associated with a final postmitotic stage.

99

~ ,~A

~

O

~ ~'

~~

.-

~

KJ~,

~. I~

~ ~ m *~ ~

~ "

~

I1~

~

~

~

~ ~

-

• ~

-

~

.

~

Fig. 18. Preferential R N P staining of nuclear material following loosening procedure. Chromatin threads are bleached whereas R N P structures are stained. The arrows indicate chromatin granules. Non-nucleolar R N P structures (arrow heads) are more frequent in 30th PDL (a) than in 59th PDL (b) cells. Cells are identical to those used for Fig. 1.

100

Fig. 19. Human fibroblasts identical to those used for Fig. 1. in phase II (A) and phase IV (B). Several works have illustrated the relationship between the evolution of morphology and the reorganization taking place in the cytoskeletal elements. Cell spreading is altered and seems to be related to a disorganization of the assembly of actin bundles (Kelley et al., 1980) and a reduction of filamin which is implicated in actin-membrane associations (Kelley et al., 1985). An increased actin content and a rearrangement of the microtubular network (Anderson, 1978; VanGansen et al., 1984; Raes et al., 1984) and of intermediate filaments (Wang, 1985) have also been reported which coincide with the appearance of the final

mitotic cell. It is pertinent to remember that actin genes seem to be affected by the genome reorganization progressing during serial proliferation of human fibroblasts (Icard-Liepkalns et al., 1986). The modifications of the cytoskeletal elements are accompanied by a decline in cell contractility which occurs in parallel with the difficulty in the initiation of DNA synthesis (Macieira-Coelho and Azzarone, 1990) and with the evolution of the alterations in the organization of the chromatin fibers at the site of their anchorage (MacieiraCoelho and Puvion-Dutilleul, 1989). Further proof of the coupling between the reorganization of the chromatin high order structure and the changes in the supporting scaffold was obtained with fibroblasts from biopsies of old donors. We found that the changes in the arrangement of the 30onm chromatin fibers are detected only in cells that migrate from the explants, in the biopsy itself one cannot see the modifications. This suggests that the cells have to attach, stretch and migrate on a substratum for the increased spacing between the solenoid fibers to become apparent; in other words, the migration and the stretching of the cells reveal the latent alterations that accumulate with senescence in chromatin. This is germane to the finding that mouse fibroblasts in collagen gels seemed stable and did not display the characteristic alterations of the cytoskeleton of an older phenotype (Leberghe and VanGansen, 1986); when the fibroblasts migrated and left the gel, the cytoskeletal elements returned to the old phenotype. A decreased cell spreading has also been obo served in fibroblasts from normal aged and Alzheimer donors (Peterson and Goldman, 1986). These works illustrate the relationship between the different constituents of this network that integrates the genome with the cytoplasm and the cell periphery; they emphasize the relevance of the flexibility of the network for the triggering of the division cycle. One can now understand a significant part of the chain of events occurring in these proliferative cell compartments that lead to a decline of the response to growth stimuli. Cell behavior depends to a great extent upon the way this network of structures integrating information within the cell is connected, i.e., its topology. The

101

genome reorganization and the decline in conformational flexibility that occur during serial divisions decrease the probability for chromatin to assume the conformational competence and initiating sites, the right steric configuration favorable for progression through the division cycle (Macieira-Coelho, 1983). These events create a drift that increases the heterogeneity of the response to growth stimuli and terminate with abrupt changes that lead to a postmitotic cell. They create new constraints that may favor the switch of cell metabolism to other functions. References Almagor, M., and R.D. Cole (1989) Changes in chromatin structure during aging of cell cultures as revealed by different scanning calorimetry, Biochemistry, 28, 56885693. Anderson, P.J. (1978) Actin in young and senescent fibroblasts, Biochem. J., 169, 169-172. Bemiller, P.M., and J.E. Miller (1979) Cytological changes in senescing WI-38 cells. A statistical analysis, Mech. Ageing Dev., 10, 1-15. Benn, P.A. (1976) Specific chromosome aberrations in senescent fibroblast cell lines derived from human embryos, Am. J. Hum. Genet., 28, 465-473. Berezney, R., and D.S. Coffey (1975) Nuclear protein matrix: association with newly synthesized DNA, Science, 189, 291-293. Bourgeois, C.A., N. Raynaud, C. Diatloff-Zito and A. Macieira-Coelho (1981) Effect of low dose rate ionizing radiation on the division potential of cells in vitro. VIII. Cytogenetic analysis of human fibroblasts, Mech. Ageing Dev., 17, 225-235. Bouvier. D., J. Hubert, A.P. Seve and M. Bouteille (1985) Characterization of lamina-bound chromatin in the nuclear shell isolated from HeLa cells, Exp. Cell Res., 156, 500-512. Brock, M.A., and R.J. Hay (1971)Comparative ultrastructure of chick fibroblasts in vitro at early and late stages during their growth span, J. Ultrastruct. Res., 36, 291-302. Chen, T.R., and F.H. Ruddle (1974) Chromosome changes revealed by the Q-band staining method during cell senescence of WI-38, Proc. Soc. Exp. Biol. Med., 147, 533-536. Comings, D.E. (1978) Compartmentalization of nuclear and chromatin protein, in: H. Busch (Ed.), The Cell Nucleus, Academic Press, New York, pp. 345-371. Crothers, D.M., T.E. Haran and J.G. Nadeau (1990) Intrinsically bent DNA, J. Biol. Chem., 265, 7093-7095. Dayton, M.A., P. Nahreini and A. Srivastava (1989) Augmented nuclease activity during cellular senescence in vitro, J. Cell. Biochem., 39, 75-85. Dell'Orco, R.T., and W.L. Whittle (1982) Micrococcal nuclease and DNase I digestion of DNA from aging human

diploid cells, Biochem. Biophys. Res. Commun., 107, 117122. Dell'Orco, R.T., and M.I. Worthington (1988) The effects of in vitro age and culture state on histone variant synthesis in human diploid fibroblasts, Biochem. Biophys. Res. Commun., 136, 168-174. Dell'Orco, R.T., W.L. Whittle and A. Macieira-Coelho (1986) Changes in the higher order organization of DNA during aging of human fibroblast-like cells, Mech. Ageing Dev., 35, 199-208. Dutkowski, R.T., R. Lesh, L. Staiano-Coico, H. Thaler, G.J. Darlington and M.E. Weksler (1985) Increased chromosomal instability in lymphocytes from elderly humans, Mutation Res., 149, 505-512. Esposito, D., G. Fassina, P. Szabo, P. De Angelis, L. Rodgers, M. Weksler and M. Siniscalco (1989) Chromosomes of older humans are more prone to aminopterine-induced breakage, Proc. Natl. Acad. Sci. (U.S.A.), 86, 1302-1306. Finch, J.T., and A. Klug (1976) Solenoid model for superstructure in chromatin, Proc. Natl. Acad. Sci. (U.S.A.), 73, 1897-1901. Flores, S.C., P. Sunnerhagen, T.K. Moore and J.W. Gaubatz (1988) Characterization of repetitive sequence families in mouse heart small polydisperse DNAs: age-related studies, Nucleic Acids Res., 16, 3889-3906. Galloway, S.M., and K.E. Buckton (1978) Aneuploidy and ageing: chromosome studies on a random sample of the population using G-banding, Cytogenet. Cell Genet., 20, 78-95. Gerace, L. (1985) Structural proteins in the eukaryotic nucleus, Nature, 318, 508-509. Harnden, D.G., P.A. Benn, J.M. Oxford, A.M.R. Taylor and T.P. Webb (1976) Cytogenetically marked clones in human fibroblasts cultured from normal subjects, Somat. Cell Genet., 2, 55-62. Hartwig., M., and I.J. K6rner (1987) Age-related changes of DNA winding and repair in human peripheral lymphocytes, Mech. Ageing Dev., 38, 73-78. Hay, M., and U. Deboni (1991) Chromatin motion in neuronal interphase nuclei - changes induced by disruption of intermediate filaments, Cell Motil. Cytoskeleton, 18, 6370. Hedner, K., B. H6gstedt, A.M. Kolnig, E. Mark-Vendel, B. Str6mbeck and F. Mitelman (1982) Sister chromatid exchanges and structural chromosome aberrations in relation to age and sex, Hum. Genet., 62, 305-309. Hill, B.T., R.D.H. Whelan and S. Whatley (1978) Evidence that transcription changes in ageing cultures are terminal events occurring after the expression of a reduced proliferative potential, Mech. Ageing Dev., 8, 85-95. Hornsby, P.J., and S.E. Harris (1987) Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells, Exp. Cell Res., 168, 203-217. Icard, C., R. Beaupain, C. Diatloff and A. Macieira-Coelho (1979) Effect of low dose rate irradiation on the division potential of cells in vitro. VI. Changes in DNA and in radiosensitivity during aging of human fibroblasts, Mech. Ageing Dev., 11,269-278.

102 Icard-Liepkalns, C., and A. Macieira-Coelho (1982) Aging and hydrocortisone effects on transient structures of replicative DNA of human fibroblasts, Proc. Soc. Exp. Med., 170, 373-377. lcard-Liepkalns, C., J. Doly and A. Macieira-Coelho (1986) Gene reorganization during serial divisions of normal human cells, Biochem. Biophys. Res. Commun., 141, 112-123. Igo-Kemenes, T. (1985) The structure of satellite-containing chromatin of the rat, in: C. Nicolini and P.O.P. Ts'o (Eds.), Structure and Function of the Genetic Apparatus, Plenum, New York, pp. 55-81. Ishimi, Y., M. Kojima, F. Takeuchi, T. Miyamato, M.-A. Yamada and F. Hanaoka (1987) Changes in chromatin structure during aging of human skin fibroblasts, Exp. Cell Res., 169, 458-467. Jackson, D.A., S.J. McCready and P.R. Cook (1984) Replication and transcription depend on attachment of DNA to the nuclear cage, J. Cell Sci., Suppl. 1, 59-79. Jacobs, P.A., W.M. Court Brown and R. Doll (1961) Distribution of human chromosome counts in relation to age, Nature, 191, 1178-1179. Johnson, R., and B.L. Strehler (1972) Loss of genes coding for ribososmal RNA in aging brain cells, Nature, 240, 412-414. Kanungo, M.S. (1980) Biochemistry of Aging, Academic Press, New York, pp. 51-78. Kelley, R.O., J.A. Trotter, L.F. Marek, B.D. Perdue and C.B. Taylor (1980) Variation in cytoskeletal assembly during spreading of progressively subcultivated human fibroblasts (IMR-90), Mech. Ageing Dev., 13, 127-141. Kelley, R.O., P.L. Mann, B.D. Perdue and L.F. Marek (1985) Reduction of filamin in late passage human diploid fibroblasts (IMR-90), Mech. Ageing Dev., 30, 78-89. Kishi, K., A. Homma, A. Kawa and K. Kadowaki (1987) Age related change in the frequency of ara-C induced chromosome aberrations in human peripheral blood lymphocytes, Mech. Ageing Dev., 37, 211-219. Kunisada, T., H. Yamagishi, Ogita, Z.-I., T. Kirakawa and Y. Mitsui (1985) Appearance of extrachromosomal circular DNAs during in vivo and in vitro ageing of mammalian cells, Mech. Ageing Dev., 29, 89-99. Lindgren, U., and J.R.A. Farber (1982) Chromosome replication in aging human diploid fibroblasts, Exp. Cell Res., 142, 301-308. Leberghe, N.V., and P. VanGansen (1986) Behaviour and ultrastructure of in vitro ageing mouse embryo fibroblasts grown in collagen, Mech. Ageing Dev., 34, 133-150. Lezhava, T.A. (1984) The activity of nucleolar organizer regions of human chromosomes in extreme old age, Gerontology, 30, 94-99. Lima, L., and A. Macieira-Coelho (1972) Parameters of aging in chicken embryo fibroblasts cultivated in vitro, Exp. Cell Res., 70, 279-284. Macieira-Coelho, A. (1979) Reorganization of the cell genome as the basis of aging in dividing cells, in: H. Orimo, K. Shimada, M. Iriki and D. Maeda (Eds.), Recent Advances in Gerontology, Excerpta Medica, Amsterdam, pp. 111112. Macieira-Coelho, A. (1980) Implications of the reorganization

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