Genomic Instability and Aging Studies in Centenarians (Successful Aging) and in Patients with Down’s Syndrome (Accelerated Aging)a CLAUD10 FRANCESCHI; DANIELA MONT1,b MARIA ROSARIA SCARF~,COLGA ZENI,C PAOLA TEMPERANI,~ GIOVANNI EMILIA: PAOLO SANSONI; MARIA BRIGIDA LIOIf LEONARDA TROIAN0,b CATERINA AGNESINI; STEFAN0 SALVIOL1,b AND ANDREA COSSARIZZAb bInstitute of General Pathology University of Modena via Campi 287 Modena 41 IOO, Italy ‘CNR-IRECE Napoli, Italy dCenterfor Experimental Hematology University of Modena Modena, Italy eMedical Clinic University of Parma Parma, Italy fDepartment of Animal Production Science University of Basilicata Potenza, Italy
THE NETWORK OF CELLULAR DEFENSE MECHANISMS
In previous papers we proposed the general hypothesis that aging and longevity are the result of a balance between mechanisms that favor aging and mechanisms that counteract the aging The mechanisms favoring aging and senescence are those that cause damage to macromolecules and other body components. They come from both exogenous and endogenous sources. Ionizing radiation, UV radiation, and xenobiotics, including dietary carcinogens, are the most common exogenous genotoxic compounds with which we cope in everyday life. Body heat, oxygen free radicals, glucose, and other oxidative sugars are representative of the byproducts of a variety of metabolic pathways and represent unavoidable, potentially genotoxic agents3
aThe data reported in this review were obtained during experiments supported by C.N.R. grants to C. Franceschi (Progetto Finalizzato “Invecchiamento,” paper no. 92-1-207, and Progetto Finalizzato “Network per la Raccolta di Materiale Biologico per Studi di Biologia Molecolare delle Malattie del Sistema Nervoso”), by grants from M.U.R.S.T. (40% and 60%), and from Regione Emilia-Romagna. 4
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Other endogenous substances that may be involved in the aging process are hormones, which can have either proaging or antiaging effects. Indeed, they are responsible for regulating developmental processes and reproduction, two phenomena deeply related to the aging proces~.~35 To maintain homeostasis, cells throughout the animal kingdom have been equipped with a variety of cellular defense mechanisms. The most important of these mechanisms are: (1) D N A repair mechanisms; (2) antioxidant systems, either enzymatic or nonenzymatic; (3) production of heat shock and other stress proteins; and (4) activation of nuclear enzymes such as poly(ADP-ribose) polymerase (PADPRP). Evidence exists that programmed cell death, o r apoptosis, may be a fifth possible cellular defense mechanism.6 W e and others argued that apoptosis can be considered one of the most important mechanisms to eliminate heavily mutated cells and thus to avoid cell t r a n s f ~ r r n a t i o nOur . ~ major assumption is that these mechanisms are interconnected and constitute a network of integrated cellular defense systems that must be considered altogether, and not one by one.' It can be assumed that the efficiency of this network is essential for the correct functioning of the three main systems responsible for body homeostasis, that is, the immune, the nervous, and the endocrine system. Owing to their strict anatomical and functional connections and to their probable common evolutionary origin, they should be considered, on the whole, as an integrated system, for which the name immunoneuroendocrine system has been suggested.s W e assume that the efficient operation of this network is the most important antiaging system of the body. According to this hypothesis, the following considerations and speculations may be put forward: 1. The efficiency of the network is genetically controlled and regulated. 2. The level and efficiency of this regulation vary among individuals of the same species as well as among different species. 3. The efficiency of the network is the major variable controlling aging and longevity. 4. Such efficiency is the result of a continuous balance between mechanisms responsible for maintaining the integrity of genetic information, on one side, and random damage to D N A and other macromolecules and pleiotropic genes controlling development and reproduction, on the ~ t h e r . ~ ~ ~ 5. The level at which the network efficiency has been set has evolved throughout evolution, so that long-living species have an extremely accurate network, whereas such accuracy is much lower in short-living species, in which the maintenance of the body is less important, owing to their higher rate of r e p r o d u c t i ~ n . ~ ~ ~ 6. The network of defense system that is mainly responsible for longevity is the same one that counteracts the natural tendency to cellular transformation and tumor incidence. Indeed, one of the main goals of the network is to preserve genetic information and to eliminate mutated molecules and cells. This may explain the apparent paradox that cellular senescence and apoptosis are the main strategies used by complex creatures to escape tumors.' HUMAN CENTENARIANS AND THE EFFICIENCY OF THE NETWORK
Long-living individuals such as centenarians are suitable for testing the hypothesis that genomic instability plays a role in the aging proces~.~-''Assuming that D N A damage accumulates with age in a constant manner, it can be expected that a variety of cytogenetic alterations have had their zenith in cells from centenarians. O n the
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other hand, a high efficiency of DNA repair mechanisms can be predicted, which should reduce the number of DNA alterations at both the molecular and the cellular levels. Accordingly, we have analyzed, with a variety of cytogenetic techniques, the genomic instability of cells from healthy centenarians. The major findings are summarized as follows: 1. Spontaneous chromatid breaks. The number of breaks per cell is considered an index of genomic instability. Indeed, alterations of this parameter are found in several diseases such as Fanconi’s syndrome, Bloom’s syndrome, scleroderma, and many forms of cancer.I2-l6 The number of spontaneous chromatid breaks is also increased in viral infections. An age-related increase of this parameter has been reported even if contradictory data exist in the l i t e r a t ~ r e . I ~ - ’ ~ The number of spontaneous chromatid breaks was measured in 9 healthy centenarians and compared with that of 25 control subjects 20-79 years old. No differencewas found between the two groups of donors, even if the number of gaps was three- to fourfold lower in centenarians than in the controls (data not shown). An increase in aneuploidy with advancing age in peripheral blood leukocytes has been reported by several authors. The utilization of chromosome banding techniques confirmed preferential X chromosome loss in hypodiploid cells of older women.20As the majority of centenarians are women, it will be of interest to check if such a preferential loss also occurs in these subjects. On the whole, these data suggest that cells from centenarians are characterized by a consistent genomic stability. 2. Bleomycin-induced chromatid breaks. The addition of DNA damaging agents such as bleomycin during the last few hours of culture allows the study of the cell sensitivity to a clastogenic agent.21 In this case, DNA is damaged, and breaks are produced in proliferating cells. These breaks, induced in G2 phase, can easily be detected in metaphase and expressed as the number of breaks per cell. The number of breaks per cell is proportional to the cell sensitivity to the damaging agent. An age-related increase in bleomycin-induced breaks per cell was observed in 25 healthy donors aged 23-79 years (FIG.1, solid line). As far as we know, this parameter has never been measured in centenarians. The number of breaks per cell observed in lymphocytes from such donors was much higher than expected (FIG.1, dotted line). This indicates that despite a basic genomic stability, centenarians’ DNA is much more sensitive to this radiomimetic agent. Indeed, in some cases a dramatic number of breaks per cell was observed after exposure to bleomycin in cells from centenarians (FIG.2, bottom). 3. Spontaneous sister chromatid exchange (SCE). SCE represents the interchange of DNA replication products at apparently homologous chromosomal loci. These exchanges presumably involve DNA breakage and reunion, and SCE analysis affords an excellent opportunity for cytological detection of DNA interchange. Abnormality in SCE formation has been described in a number of heritable human diseases that are characterized by a putative DNA repair defect and a predisposition to the development of neoplasia.22 SCE is also increased in workers exposed to putative carcinogenetic agentsz3The widespread occurrence of SCE represents an important clue in the investigation of structure-function relationships in the chromosomes of higher organisms, and the analysis of their frequency is considered to be a rapid and sensitive method for the quantification of DNA damage. Although the biological significance and molecular mechanisms involved in SCE formation are still poorly understood, SCE may represent a chromosomal response to DNA damage at the time of DNA replication. Contradictory data exist in the literature concerning the age-related increase of SCE.24*25 Centenarians represent a selected population of persons who escaped major age-related disease, including neoplasia. As tumors are
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the result of several mutagenic events, the extremely low frequency of cancer in centenarians suggests that their cells are equipped with efficient antimutagenic mechanisms.To test this hypothesis, the spontaneous frequency of SCE was assessed in these subjects and in healthy controls of different ages. FIGURE 3 shows that the frequency of SCE per cell significantly increases with age ( p < 0.025) in 25 people 23-79 years old (solid line). One could expect that the frequency of spontaneous SCE per cell would be even higher in people over 80. In particular, according to this extrapolation, the expected values for centenarians should be about eight SCEs per cell (FIG.3, dotted line). O n the contrary, the SCE frequency measured in nine centenarians was significantly lower than expected (FIG. 3, open circles). Thus, according to another cytogenetic test that measures the capability of the genome to recombine, it is evident that centenarians have a lower
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AGE (years) FIGURE 1. Age-related increase of bleomycin (25 kg/rnl, last 5 hours of culture)-induced chromatid breaks in human peripheral blood lymphocytes. Data are expressed as breaks per cell. The solid line represents the regression line of the experimental values obtained in our laboratory on 25 healthy donors of different ages (23-79 years), and the dotted line represents its theoretical extrapolation after the age of 80 years. Each open circle refers to a centenarian.
tendency to exchange genetic material and a high genomic stability. Two representative cells from centenarians showing relatively low and high number of SCEs per cell 4. are shown in FIGURE 4. Spontaneous and mitomycin-C (MMC)-induced micronuclei (MN). Micronuclei indicate the presence of acentric chromosome fragments o r whole chromosomes that have not been incorporated in the main nucleus at cell division. The cytokinesisblock MN method, introduced by Fenech and Morley,2hwas used in our experiments. This is a simple and sensitive method of scoring chromosomal damage in cells that have undergone one cell division after the clastogenic insult.27 Moreover, this technique is more sensitive than the conventional MN method and classical chromosomal aberration assays. Indeed, a strong correlation between chromosomal aberrations and MN formation has been shown2*The frequency of MN can be assessed in
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FIGURE 2. Two representative examples of metaphases showing bleomycin-induced breaks observed. The data refer to two centenarians. The upper and lowerpafls of the figure show cells with a low and a high number of breaks (arrows),respectively.
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either the absence or the presence of genotoxic agents, and these parameters are indicated as spontaneous or induced MN, respectively. An increase in spontaneous MN frequency with age has been reported.26 The spontaneous and MMC-induced MN frequency in cells from centenarians and healthy subjects of different ages was 5 shows that an age-related increase in the spontaneous MN assessed. FIGURE frequency exists in 35 donors aged 20-95 years (solid line, a). The age-related increase in spontaneous MN frequency, although statistically significant, is lower than that reported by other authorsz6 As far as we know, the MN frequency in people over 80 has never been reported. However, such frequency can be extrapolated assuming that the rate of the phenomenon remain constant with age (FIG.5, dotted line, a). Indeed, the spontaneous MN frequency observed in cells from six centenarians is apparently higher than expected (FIG.5, open circles).
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AGE (years) FIGURE 3. Age-related increase of the spontaneous number of sister chromatid exchanges (SCE) in human peripheral blood lymphocytes (BrdU 10 pg/ml for 36 hours). Data are expressed as the number of SCEs per cell. The solid line represents the regression line of the experimental values obtained in our laboratory on 25 healthy donors of different ages (23-79 years), and the dotted line represents its theoretical extrapolation after the age of 80 years. Each open circle refers to a centenarian.
The MN frequency is usually highly increased if cells are cultured in the presence of a clastogenic agent. In a previous paper we showed that in healthy adults the MN frequency doubled after MMC treatment.29The study of 35 subjects of different ages showed that the frequency of MMC-induced MN does not change with age (FIG.5, solid line, b). This is also true for the six centenarians studied, in whom the high spontaneous MN frequency does not change in MMC-treated cells (FIG. 5, black circles). This is in agreement with previous observations showing that MN frequency did not change with age after X-ray exposure.3oIn any case, it is interesting to note that in cells from centenarians the MMC-induced MN frequency was much higher than expected (FIG.5, dotted line, b).
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FIGUREI. Two representative examplesof cells with SCE. The data refer to two centenarians. The upper and lowerpurts of the figure show cells with a high and a low number of SCEs (arrows), respectively.
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SYNDROMES OF PRECOCIOUS AGING AND THE FAILURE OF THE NETWORK Down's syndrome (DS) ranks at the top among human segmental progeroid syndromes, defined as those genetic disorders in which multiple major aspects of the senescent phenotype appear.3LIn other words, it has been shown that most features of physiological aging appear in DS earlier than in karyotypically normal agematched subjects. Apart from a decreased life expectancy and an accelerated death rate beginning by about age 40, many progeroid characteristics have been reported in DS, including cataracts, alopecia, premature greying of hair, hypogonadism, presby-
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AGE (YEARS) FIGURE 5. Age-related increase of the spontaneous (a) and mitomycin-C (MMC)-induced (b) micronuclei (MN) frequency in binucleated cells from centenarians. The solid lines (a and b) represent the regression lines of the experimental values obtained in our laboratory on 35 healthy donors of different ages (2&95 years), and the dotted lines their theoretical extrapolation after the age of 95 years. For experimental details, see ref. 29.
acusis, akin atrophy, diabetes mellitus, increased prevalence of amyloidosis, neurofibrillary tangles, and increased susceptibility to leukemia. Changes similar to those of normal aging seem to occur in DS brain some 20 years before they do in controls, as suggested by recent studies on P300 latency. Moreover, a disease similar to Alzheimer's disease appears to be present in DS patients dying after 40. Almost 100% of DS subjects (either trisomy 21 or partial trisomy 21) over 40 years suffer from brain histopathological changes similar to those found in Alzheimer's disease (senile plaques and neurofibrillary tangles). D N A repair defects have been described in DS.32 The presence of genomic instability in this syndrome has been d i s c ~ s s e d . ~ '
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DS subjects had a lower frequency of spontaneous MN (FIG. 6, line a) and an increased frequency of MMC-induced MN (FIG. 6, line b) than did control donors owing to a dramatic age-dependent effect. The proliferative capability of old DS subjects was much lower than that of age-matched controls (FIG. 7). However, the cytokinesis block method that we have used to assess the MN frequency should reasonably exclude variations in the lymphocyte response to PHA.26,33In this way, differences between the proliferative capability of lymphocytes from DS and control subjects and from young and aged DS subjects should be minimized. These data suggest that a genomic instability is likely present in DS, but this instability appears to be age-dependent, becoming evident only in aged DS subjects. A similar age-related phenomenon occurs when lymphocytes from DS patients are exposed to extremely low-frequency pulsed electromagnetic fields ( P E M F s ) . ~In~ deed, cells from adult DS subjects showed a sensitivity to PEMFs similar to that found in normal aged donors. However, the genomic instability likely present in DS is apparently different from that present in physiological aging. Indeed, an agerelated increase in spontaneous MN frequency was found in karyotypically normal donors. However, no difference between the level of X-ray30 and MMC-induced (present paper) MN in young and old normal subjects was found. The reason for this apparent discrepancy between spontaneous and induced MN frequency in normal aging and in DS is presently unclear. In general, when comparing sensitivity to genotoxic agents in DS and normal subjects, the unsuspected age-related effect we have described must be taken into account. The lack of evidence for genomic instability in cells from young DS subjects does not exclude the possibility that such a defect might be detected when cells from older DS donors are studied.
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AGE (YEARS) FIGURE 6. Age-related increase of the spontaneous (a) and mitomycin-C (MMC)-induced (b) micronuclei (MN) frequency in binucleated cells from subjects affected by Down’s syndrome. Lines a and b represent the regression lines of the reported data (from Ref. 29).
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PHA (pl/ml) FIGURE 7. Phytohemagglutinin (PHA)-induced lymphocyte proliferation of peripheral blood lymphocytes from six patients affected by Down's syndrome and from six age- and sex-matched controls. Data represent the ('H)-thymidine incorporation during the last 6 hours of a 72-hour culture and are expressed as counts per minute (cpm) (mean 2 SEM). For experimental details see ref. 34.
CONCLUSIONS The general hypothesis that persons who have been able to reach thc extreme limit of human lifespan could represent a suitable model to assess the importance of genomic stability/instability in the aging process was evaluated by using a variety of cytogenetic techniques. Two biological models were studied, that is, centenarians as an example of successful aging, and subjects with Down's syndrome, a situation in which most of the major signs of the aging process appear precociously. The hypothesis that genomic instability increases with age and that genomic stability is a prerequisite for longevity is likely an oversimplification. It is clear that increased genomic stability or instability can be claimed according to the method used. Increascd or decreased sensitivity to genotoxic agcnts such as bleomycin and mitomycin-C, respectively, is apparently present in centenarians. The significance of this phenomenon is presently unclear. The low number of spontaneous chromatid breaks recorded in cells from centenarians suggests that heavily damaged cells are efficiently repaired or eliminated. A high capability to repair DNA damages can bc predicted in cells from centenarians. This prediction is in agreement with previous data from our laboratory, which showed that pcripheral blood mononuclear cells from centenarians can efficiently cope with oxidative stress.3* Alternatively, it can be hypothesized that cells from centenarians are more prone to programmed cell death, a mechanism that could help the body to eliminate potentially dangerous (cell
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transformation), heavily damaged/mutated c e k 7In any case, we have to explain the fact that healthy centenarians escaped from major age-related diseases including cancer, a process in which several mutational events are required and that is highly facilitated by genomic instability. The other model we have chosen to assess the importance of genomic stability/ instability in the aging process, that is, Down’s syndrome, also gave an intriguing answer to the question. Indeed, a complex, and at present unexplained, age-related increased sensitivity to genotoxic agents is present in this syndrome. Most previous data of the literature on the frequency of SCE or chromosome breaks did not take into account the importance of the patient’s age. Thus, we are in a situation in which more data are needed in DS, particularly a careful analysis of genomic instability with age. The hypothesis can be suggested that this age-related increased sensitivity to genotoxic substances can contribute to the acceleration of the aging process, particularly evident in DS subjects older than 30 years. The question of the importance of genomic stability/instability in the aging process will likely be properly answered when more precise and sophisticated techniques for the assessment of DNA damage and repair in single cells and at the nucleotide level are extensively used. However, the work to be done is tremendous, taking into account that different cells of different tissues and organs can be differently affected by the aging process and that a continuous balance between phenomena that tend to destabilize DNA and other macromolecules, and phenomena that repair macromolecules and eliminate damaged cells continuously takes place in human body. In previous studies we showed that centenarians have very efficient immune response^^^.^^-^^ and that, on the contrary, an age-related deterioration of the immune system occurs in DS subjects.3w2 These features are likely of major importance in explaining successful and precocious aging, respectively. One of the major efforts of our laboratory is to ascertain if in aging and longevity a relation exists between the efficiency or the impairment of the immune response and alterations in the mechanisms responsible for the maintenance of genomic integrity. ACKNOWLEDGMENT
We thank Professor M. Varricchio, Department of Gerontology, University of Napoli, for his help in blood collection. REFERENCES
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ANNALS NEW YORK ACADEMY OF SCIENCES exchanges induced by trimethyltin chloride in human peripheral blood lymphocytes as related to age of donors. A brief report. Mech. Ageing Dev. 5 0 95-102. FENECH,M. & A. A. MORLEY.1986. Cytokinesis-block micronucleus method in human lymphocytes: Effect of in vivo ageing and low dose X-irradiation. Mutat. Res. 161: 193198. HUBER,R., H. BRASELMANN & M. BAUCHINGER. 1992. Intra- and inter-individual variation of background and radiation-induced micronucleus frequency in human lymphocytes. Int. J. Radiat. Biol. 61: 655-661. RAMALHO, A,, I. SUNJEVARIC & A. T. NATARJAN.1988. Use of the frequencies of micronuclei as quantitative indicators of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes: Comparison of two methods. Mutat. Res. 207: 141-146. SCARFI,M. R., A. COSSARIZZA, D. MONTI,F. BERSANI,M. ZANNOTTI, M. B. LIOI& C. FRANCESCHI. 1990. Age-related increase of mitomycin-C-induced micronuclei in lymphocytes from Down’s syndrome subjects. Mutat. Res. 237: 217-222. FENECH,M. & A. A. MORLEY.1987. Ageing in vivo does not influence micronucleus induction in human lymphocytes by X-irradiation. Mech. Ageing Dev. 3 9 113-119. FRANCESCHI, C., F. LICASTRO, M. CHIRICOLO, M. ZANNOTTI & M. MASI.1986. Premature senility in Down’s syndrome: A model for and an approach to the molecular genetics of the ageing process. In Immunoregulation in Ageing. A Facchini, J. J. Haaijman & G. Labo, eds.: 77-83. Eurage Pub]., Rijswijk, The Netherlands. G. PASSERI & P. SANSONI. 1991. FRANCESCHI, C., D. MONTI,A. COSSARIZZA, F. FAGNONI, Aging, longevity and cancer: Studies in Down’s syndrome and centenarians. Ann. N.Y. Acad. Sci. 621: 428-441. FENECH,M. & A. A. MORLEY.1985. The effect of donor age on spontaneous and induced micronuclei. Mutat. Res. 148 99-105. COSSARIZZA, A,, D. MONTI,F. BERSANI, M. R. SCARF^, M. ZANNOTTI, R. CADOSSI& C. 1991. Extremely low frequency pulsed electromagnetic fields increase FRANCESCHI. lymphocyte proliferation in Down’s syndrome. Aging 3: 241-246. MARIOTTI, S., P. SANSONI, G. BARBESINO, P. CATUREGLI, D. MONTI,A. COSSARIZZA, T. GIACOMELLI, U. FAGIOLO,A. PINCHERA & C. FRANCESCHI. 1992. Thyroid and other organ-specific autoantibodies in healthy centenarians. Lancet 339 1506-1508. COSSARIZZA, A., C. ORTOLANI, R. PAGANELLI, D. MONTI,D. BARBIERI, P. SANSONI, U. FAGIOLO,E. FORTI,M. LONDEI& C. FRANCESCHI. 1992. Age-related unbalance of virgin (CD45RA+) and memory (CD45RO+) cells between CD4+ and CD8+ T lymphocytes in humans: A study from newborns to centenarians. J. Immunol. Res. 4 118-126. A. COSSARIZZA, C. ORTOLANI, E. GUERRA,P. PAGANELLI, R., I. QUINTI,U. FAGIOLO, 1992. SANSONI, L. P. PUCILLO, E. COZZI,L. BERTOLLO, D. MONTI& C. FRANCESCHI. Circulating B cells and Ig classes and subclasses in healthy aged people and in centenarians. Clin. Exp. Immunol. (In press). & M. ZANNOTTI. 1978. FRANCESCHI, C., F. LICASTRO, P. PAOLUCCI, M. MASI,S. CAVICCHI T and B lymphocyte subpopulations in Down’s syndrome. Study on not-institutionalized subjects. J. Ment. Defic. Res. 22: 179-181. FRANCESCHI, C., F. LICASTRO, M. CHIRICOLO, F. BONETTI,M. ZANNOTTI, N. FABRIS,E. & M. MASI.1981. Deficiency of autologous MOCCHEGIANI, M. P. FANTINI,P. PAOLUCCI mixed lymphocyte reactions and serum thymic factor level in Down’s syndrome. J. Immunol. 126 2161-2164. FABRIS,N., E. MOCCHEGIANI, L. AMADIO, M. ZANNOTTI, F. LICASTRO & C. FRANCESCHI. 1984. Thymic hormone deficiency in normal ageing and Down’s syndrome: Is there a primary failure of the thymus? Lancet i: 983-986. COSSARIZZA, A,, D. MONTI,G. MONTAGNANI, C. ORTOLANI, M. MASI,M. ZANNOTTI & C. FRANCESCHI. 1990. Precocious aging of the immune system in Down’s syndrome: Alterations of B-lymphocytes, T-lymphocyte subsets and of cells with NK markers in DS children. Am. J. Med Genet. 7 (suppl): 213-218. COSSARIZZA, A., C. ORTOLANI, E. FORTI, G . MONTAGNANI, R. PAGANELLI, M. ZANNOTTI, 1991. Age-related expansion of functionally M. MARINI,D. MONTI& C. FRANCESCHI. inefficient cells with markers of NK activity in Down’s syndrome. Blood 77: 1263-1270.
THE NETWORK OF CELLULAR DEFENSE MECHANISMS
In previous papers we proposed the general hypothesis that aging and longevity are the result of a balance between mechanisms that favor aging and mechanisms that counteract the aging The mechanisms favoring aging and senescence are those that cause damage to macromolecules and other body components. They come from both exogenous and endogenous sources. Ionizing radiation, UV radiation, and xenobiotics, including dietary carcinogens, are the most common exogenous genotoxic compounds with which we cope in everyday life. Body heat, oxygen free radicals, glucose, and other oxidative sugars are representative of the byproducts of a variety of metabolic pathways and represent unavoidable, potentially genotoxic agents3
aThe data reported in this review were obtained during experiments supported by C.N.R. grants to C. Franceschi (Progetto Finalizzato “Invecchiamento,” paper no. 92-1-207, and Progetto Finalizzato “Network per la Raccolta di Materiale Biologico per Studi di Biologia Molecolare delle Malattie del Sistema Nervoso”), by grants from M.U.R.S.T. (40% and 60%), and from Regione Emilia-Romagna. 4
FRANCESCHI el al.: GENOMIC INSTABILITY AND AGING
5
Other endogenous substances that may be involved in the aging process are hormones, which can have either proaging or antiaging effects. Indeed, they are responsible for regulating developmental processes and reproduction, two phenomena deeply related to the aging proces~.~35 To maintain homeostasis, cells throughout the animal kingdom have been equipped with a variety of cellular defense mechanisms. The most important of these mechanisms are: (1) D N A repair mechanisms; (2) antioxidant systems, either enzymatic or nonenzymatic; (3) production of heat shock and other stress proteins; and (4) activation of nuclear enzymes such as poly(ADP-ribose) polymerase (PADPRP). Evidence exists that programmed cell death, o r apoptosis, may be a fifth possible cellular defense mechanism.6 W e and others argued that apoptosis can be considered one of the most important mechanisms to eliminate heavily mutated cells and thus to avoid cell t r a n s f ~ r r n a t i o nOur . ~ major assumption is that these mechanisms are interconnected and constitute a network of integrated cellular defense systems that must be considered altogether, and not one by one.' It can be assumed that the efficiency of this network is essential for the correct functioning of the three main systems responsible for body homeostasis, that is, the immune, the nervous, and the endocrine system. Owing to their strict anatomical and functional connections and to their probable common evolutionary origin, they should be considered, on the whole, as an integrated system, for which the name immunoneuroendocrine system has been suggested.s W e assume that the efficient operation of this network is the most important antiaging system of the body. According to this hypothesis, the following considerations and speculations may be put forward: 1. The efficiency of the network is genetically controlled and regulated. 2. The level and efficiency of this regulation vary among individuals of the same species as well as among different species. 3. The efficiency of the network is the major variable controlling aging and longevity. 4. Such efficiency is the result of a continuous balance between mechanisms responsible for maintaining the integrity of genetic information, on one side, and random damage to D N A and other macromolecules and pleiotropic genes controlling development and reproduction, on the ~ t h e r . ~ ~ ~ 5. The level at which the network efficiency has been set has evolved throughout evolution, so that long-living species have an extremely accurate network, whereas such accuracy is much lower in short-living species, in which the maintenance of the body is less important, owing to their higher rate of r e p r o d u c t i ~ n . ~ ~ ~ 6. The network of defense system that is mainly responsible for longevity is the same one that counteracts the natural tendency to cellular transformation and tumor incidence. Indeed, one of the main goals of the network is to preserve genetic information and to eliminate mutated molecules and cells. This may explain the apparent paradox that cellular senescence and apoptosis are the main strategies used by complex creatures to escape tumors.' HUMAN CENTENARIANS AND THE EFFICIENCY OF THE NETWORK
Long-living individuals such as centenarians are suitable for testing the hypothesis that genomic instability plays a role in the aging proces~.~-''Assuming that D N A damage accumulates with age in a constant manner, it can be expected that a variety of cytogenetic alterations have had their zenith in cells from centenarians. O n the
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other hand, a high efficiency of DNA repair mechanisms can be predicted, which should reduce the number of DNA alterations at both the molecular and the cellular levels. Accordingly, we have analyzed, with a variety of cytogenetic techniques, the genomic instability of cells from healthy centenarians. The major findings are summarized as follows: 1. Spontaneous chromatid breaks. The number of breaks per cell is considered an index of genomic instability. Indeed, alterations of this parameter are found in several diseases such as Fanconi’s syndrome, Bloom’s syndrome, scleroderma, and many forms of cancer.I2-l6 The number of spontaneous chromatid breaks is also increased in viral infections. An age-related increase of this parameter has been reported even if contradictory data exist in the l i t e r a t ~ r e . I ~ - ’ ~ The number of spontaneous chromatid breaks was measured in 9 healthy centenarians and compared with that of 25 control subjects 20-79 years old. No differencewas found between the two groups of donors, even if the number of gaps was three- to fourfold lower in centenarians than in the controls (data not shown). An increase in aneuploidy with advancing age in peripheral blood leukocytes has been reported by several authors. The utilization of chromosome banding techniques confirmed preferential X chromosome loss in hypodiploid cells of older women.20As the majority of centenarians are women, it will be of interest to check if such a preferential loss also occurs in these subjects. On the whole, these data suggest that cells from centenarians are characterized by a consistent genomic stability. 2. Bleomycin-induced chromatid breaks. The addition of DNA damaging agents such as bleomycin during the last few hours of culture allows the study of the cell sensitivity to a clastogenic agent.21 In this case, DNA is damaged, and breaks are produced in proliferating cells. These breaks, induced in G2 phase, can easily be detected in metaphase and expressed as the number of breaks per cell. The number of breaks per cell is proportional to the cell sensitivity to the damaging agent. An age-related increase in bleomycin-induced breaks per cell was observed in 25 healthy donors aged 23-79 years (FIG.1, solid line). As far as we know, this parameter has never been measured in centenarians. The number of breaks per cell observed in lymphocytes from such donors was much higher than expected (FIG.1, dotted line). This indicates that despite a basic genomic stability, centenarians’ DNA is much more sensitive to this radiomimetic agent. Indeed, in some cases a dramatic number of breaks per cell was observed after exposure to bleomycin in cells from centenarians (FIG.2, bottom). 3. Spontaneous sister chromatid exchange (SCE). SCE represents the interchange of DNA replication products at apparently homologous chromosomal loci. These exchanges presumably involve DNA breakage and reunion, and SCE analysis affords an excellent opportunity for cytological detection of DNA interchange. Abnormality in SCE formation has been described in a number of heritable human diseases that are characterized by a putative DNA repair defect and a predisposition to the development of neoplasia.22 SCE is also increased in workers exposed to putative carcinogenetic agentsz3The widespread occurrence of SCE represents an important clue in the investigation of structure-function relationships in the chromosomes of higher organisms, and the analysis of their frequency is considered to be a rapid and sensitive method for the quantification of DNA damage. Although the biological significance and molecular mechanisms involved in SCE formation are still poorly understood, SCE may represent a chromosomal response to DNA damage at the time of DNA replication. Contradictory data exist in the literature concerning the age-related increase of SCE.24*25 Centenarians represent a selected population of persons who escaped major age-related disease, including neoplasia. As tumors are
I
FRANCESCHI et nl.: GENOMIC INSTABILITY AND AGING
the result of several mutagenic events, the extremely low frequency of cancer in centenarians suggests that their cells are equipped with efficient antimutagenic mechanisms.To test this hypothesis, the spontaneous frequency of SCE was assessed in these subjects and in healthy controls of different ages. FIGURE 3 shows that the frequency of SCE per cell significantly increases with age ( p < 0.025) in 25 people 23-79 years old (solid line). One could expect that the frequency of spontaneous SCE per cell would be even higher in people over 80. In particular, according to this extrapolation, the expected values for centenarians should be about eight SCEs per cell (FIG.3, dotted line). O n the contrary, the SCE frequency measured in nine centenarians was significantly lower than expected (FIG. 3, open circles). Thus, according to another cytogenetic test that measures the capability of the genome to recombine, it is evident that centenarians have a lower
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AGE (years) FIGURE 1. Age-related increase of bleomycin (25 kg/rnl, last 5 hours of culture)-induced chromatid breaks in human peripheral blood lymphocytes. Data are expressed as breaks per cell. The solid line represents the regression line of the experimental values obtained in our laboratory on 25 healthy donors of different ages (23-79 years), and the dotted line represents its theoretical extrapolation after the age of 80 years. Each open circle refers to a centenarian.
tendency to exchange genetic material and a high genomic stability. Two representative cells from centenarians showing relatively low and high number of SCEs per cell 4. are shown in FIGURE 4. Spontaneous and mitomycin-C (MMC)-induced micronuclei (MN). Micronuclei indicate the presence of acentric chromosome fragments o r whole chromosomes that have not been incorporated in the main nucleus at cell division. The cytokinesisblock MN method, introduced by Fenech and Morley,2hwas used in our experiments. This is a simple and sensitive method of scoring chromosomal damage in cells that have undergone one cell division after the clastogenic insult.27 Moreover, this technique is more sensitive than the conventional MN method and classical chromosomal aberration assays. Indeed, a strong correlation between chromosomal aberrations and MN formation has been shown2*The frequency of MN can be assessed in
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FIGURE 2. Two representative examples of metaphases showing bleomycin-induced breaks observed. The data refer to two centenarians. The upper and lowerpafls of the figure show cells with a low and a high number of breaks (arrows),respectively.
FRANCESCHI el al.: GENOMIC INSTABILITY AND AGING
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either the absence or the presence of genotoxic agents, and these parameters are indicated as spontaneous or induced MN, respectively. An increase in spontaneous MN frequency with age has been reported.26 The spontaneous and MMC-induced MN frequency in cells from centenarians and healthy subjects of different ages was 5 shows that an age-related increase in the spontaneous MN assessed. FIGURE frequency exists in 35 donors aged 20-95 years (solid line, a). The age-related increase in spontaneous MN frequency, although statistically significant, is lower than that reported by other authorsz6 As far as we know, the MN frequency in people over 80 has never been reported. However, such frequency can be extrapolated assuming that the rate of the phenomenon remain constant with age (FIG.5, dotted line, a). Indeed, the spontaneous MN frequency observed in cells from six centenarians is apparently higher than expected (FIG.5, open circles).
0
AGE (years) FIGURE 3. Age-related increase of the spontaneous number of sister chromatid exchanges (SCE) in human peripheral blood lymphocytes (BrdU 10 pg/ml for 36 hours). Data are expressed as the number of SCEs per cell. The solid line represents the regression line of the experimental values obtained in our laboratory on 25 healthy donors of different ages (23-79 years), and the dotted line represents its theoretical extrapolation after the age of 80 years. Each open circle refers to a centenarian.
The MN frequency is usually highly increased if cells are cultured in the presence of a clastogenic agent. In a previous paper we showed that in healthy adults the MN frequency doubled after MMC treatment.29The study of 35 subjects of different ages showed that the frequency of MMC-induced MN does not change with age (FIG.5, solid line, b). This is also true for the six centenarians studied, in whom the high spontaneous MN frequency does not change in MMC-treated cells (FIG. 5, black circles). This is in agreement with previous observations showing that MN frequency did not change with age after X-ray exposure.3oIn any case, it is interesting to note that in cells from centenarians the MMC-induced MN frequency was much higher than expected (FIG.5, dotted line, b).
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FIGUREI. Two representative examplesof cells with SCE. The data refer to two centenarians. The upper and lowerpurts of the figure show cells with a high and a low number of SCEs (arrows), respectively.
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SYNDROMES OF PRECOCIOUS AGING AND THE FAILURE OF THE NETWORK Down's syndrome (DS) ranks at the top among human segmental progeroid syndromes, defined as those genetic disorders in which multiple major aspects of the senescent phenotype appear.3LIn other words, it has been shown that most features of physiological aging appear in DS earlier than in karyotypically normal agematched subjects. Apart from a decreased life expectancy and an accelerated death rate beginning by about age 40, many progeroid characteristics have been reported in DS, including cataracts, alopecia, premature greying of hair, hypogonadism, presby-
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AGE (YEARS) FIGURE 5. Age-related increase of the spontaneous (a) and mitomycin-C (MMC)-induced (b) micronuclei (MN) frequency in binucleated cells from centenarians. The solid lines (a and b) represent the regression lines of the experimental values obtained in our laboratory on 35 healthy donors of different ages (2&95 years), and the dotted lines their theoretical extrapolation after the age of 95 years. For experimental details, see ref. 29.
acusis, akin atrophy, diabetes mellitus, increased prevalence of amyloidosis, neurofibrillary tangles, and increased susceptibility to leukemia. Changes similar to those of normal aging seem to occur in DS brain some 20 years before they do in controls, as suggested by recent studies on P300 latency. Moreover, a disease similar to Alzheimer's disease appears to be present in DS patients dying after 40. Almost 100% of DS subjects (either trisomy 21 or partial trisomy 21) over 40 years suffer from brain histopathological changes similar to those found in Alzheimer's disease (senile plaques and neurofibrillary tangles). D N A repair defects have been described in DS.32 The presence of genomic instability in this syndrome has been d i s c ~ s s e d . ~ '
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DS subjects had a lower frequency of spontaneous MN (FIG. 6, line a) and an increased frequency of MMC-induced MN (FIG. 6, line b) than did control donors owing to a dramatic age-dependent effect. The proliferative capability of old DS subjects was much lower than that of age-matched controls (FIG. 7). However, the cytokinesis block method that we have used to assess the MN frequency should reasonably exclude variations in the lymphocyte response to PHA.26,33In this way, differences between the proliferative capability of lymphocytes from DS and control subjects and from young and aged DS subjects should be minimized. These data suggest that a genomic instability is likely present in DS, but this instability appears to be age-dependent, becoming evident only in aged DS subjects. A similar age-related phenomenon occurs when lymphocytes from DS patients are exposed to extremely low-frequency pulsed electromagnetic fields ( P E M F s ) . ~In~ deed, cells from adult DS subjects showed a sensitivity to PEMFs similar to that found in normal aged donors. However, the genomic instability likely present in DS is apparently different from that present in physiological aging. Indeed, an agerelated increase in spontaneous MN frequency was found in karyotypically normal donors. However, no difference between the level of X-ray30 and MMC-induced (present paper) MN in young and old normal subjects was found. The reason for this apparent discrepancy between spontaneous and induced MN frequency in normal aging and in DS is presently unclear. In general, when comparing sensitivity to genotoxic agents in DS and normal subjects, the unsuspected age-related effect we have described must be taken into account. The lack of evidence for genomic instability in cells from young DS subjects does not exclude the possibility that such a defect might be detected when cells from older DS donors are studied.
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AGE (YEARS) FIGURE 6. Age-related increase of the spontaneous (a) and mitomycin-C (MMC)-induced (b) micronuclei (MN) frequency in binucleated cells from subjects affected by Down’s syndrome. Lines a and b represent the regression lines of the reported data (from Ref. 29).
FRANCESCHI et al.: GENOMIC INSTABILITY AND AGING
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Q
Y
PHA (pl/ml) FIGURE 7. Phytohemagglutinin (PHA)-induced lymphocyte proliferation of peripheral blood lymphocytes from six patients affected by Down's syndrome and from six age- and sex-matched controls. Data represent the ('H)-thymidine incorporation during the last 6 hours of a 72-hour culture and are expressed as counts per minute (cpm) (mean 2 SEM). For experimental details see ref. 34.
CONCLUSIONS The general hypothesis that persons who have been able to reach thc extreme limit of human lifespan could represent a suitable model to assess the importance of genomic stability/instability in the aging process was evaluated by using a variety of cytogenetic techniques. Two biological models were studied, that is, centenarians as an example of successful aging, and subjects with Down's syndrome, a situation in which most of the major signs of the aging process appear precociously. The hypothesis that genomic instability increases with age and that genomic stability is a prerequisite for longevity is likely an oversimplification. It is clear that increased genomic stability or instability can be claimed according to the method used. Increascd or decreased sensitivity to genotoxic agcnts such as bleomycin and mitomycin-C, respectively, is apparently present in centenarians. The significance of this phenomenon is presently unclear. The low number of spontaneous chromatid breaks recorded in cells from centenarians suggests that heavily damaged cells are efficiently repaired or eliminated. A high capability to repair DNA damages can bc predicted in cells from centenarians. This prediction is in agreement with previous data from our laboratory, which showed that pcripheral blood mononuclear cells from centenarians can efficiently cope with oxidative stress.3* Alternatively, it can be hypothesized that cells from centenarians are more prone to programmed cell death, a mechanism that could help the body to eliminate potentially dangerous (cell
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transformation), heavily damaged/mutated c e k 7In any case, we have to explain the fact that healthy centenarians escaped from major age-related diseases including cancer, a process in which several mutational events are required and that is highly facilitated by genomic instability. The other model we have chosen to assess the importance of genomic stability/ instability in the aging process, that is, Down’s syndrome, also gave an intriguing answer to the question. Indeed, a complex, and at present unexplained, age-related increased sensitivity to genotoxic agents is present in this syndrome. Most previous data of the literature on the frequency of SCE or chromosome breaks did not take into account the importance of the patient’s age. Thus, we are in a situation in which more data are needed in DS, particularly a careful analysis of genomic instability with age. The hypothesis can be suggested that this age-related increased sensitivity to genotoxic substances can contribute to the acceleration of the aging process, particularly evident in DS subjects older than 30 years. The question of the importance of genomic stability/instability in the aging process will likely be properly answered when more precise and sophisticated techniques for the assessment of DNA damage and repair in single cells and at the nucleotide level are extensively used. However, the work to be done is tremendous, taking into account that different cells of different tissues and organs can be differently affected by the aging process and that a continuous balance between phenomena that tend to destabilize DNA and other macromolecules, and phenomena that repair macromolecules and eliminate damaged cells continuously takes place in human body. In previous studies we showed that centenarians have very efficient immune response^^^.^^-^^ and that, on the contrary, an age-related deterioration of the immune system occurs in DS subjects.3w2 These features are likely of major importance in explaining successful and precocious aging, respectively. One of the major efforts of our laboratory is to ascertain if in aging and longevity a relation exists between the efficiency or the impairment of the immune response and alterations in the mechanisms responsible for the maintenance of genomic integrity. ACKNOWLEDGMENT
We thank Professor M. Varricchio, Department of Gerontology, University of Napoli, for his help in blood collection. REFERENCES
C. 1989. Cell proliferation, cell death and aging. Aging 1: 1-13. 1. FRANCESCHI, C . & D. MONTI. 1990. 11 controllo genetic0 dell’invecchiamento e della 2. FRANCESCHI, longeviti: Peculiariti e paradossi. Giorn. Gerontol. 3 8 127-134. C., A. COSSARIZZA, D. MONTI,V. COMASCHI, G. FARRUGGIA, G. SARTOR& 3. FRANCESCHI, L. MASOITI.1990. Cellular defense mechanisms, cell death and ageing: An integrated view. In Modern Aging Research, Volume 9, Protein Metabolism in Aging. H.L. Segal, M. Rothstein & E. Bergamini, eds.: 381-386. Wiley-Liss Inc., New York. 4. KIRKWOOD, T. B. L. & M. R. ROSE.1991. Evolution of senescence: Late survival sacrificed for reproduction. Phil. Trans. R. SOC.Lond. B. 332: 15-24. 5 . KIRKWOOD, T. B. L. & C. FRANCESCHI. 1992. Is aging as complex as it would appear? New perspectives in aging research. Ann. N.Y. Acad. Sci. This volume. D., L. TROIANO, F. TROPEA,E. GuSSILLI, A. COSSARIZZA, D. BAROZZI,M. C. 6. MONTI,
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7.
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