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Somatic mutations and cellular aging
Comp. Biochem. Physiol. Vol. 104B,No. 3, pp. 429--437, 1993
0305-0491/93 $6.00+0.00 © 1993Pergamon Press Ltd
Printed in Great Britain
M I N I REVIEW
SOMATIC MUTATIONS AND CELLULAR AGING J. VIJG and J. A. GossEr~ Harvard Medical School, Division on Aging, 643 Huntington Avenue, Boston, MA 02115, U.S.A.; and Ingeny B.V., P.O. Box 685, 2300 Ar Leiden, The Netherlands (Fax 31 71 210236) (Received 7 October 1992; accepted I 1 November 1992)
Changes in the D N A information content are fundamental to the continuous emergence of new life forms through evolution by natural selection. In multicellular organisms, however, the same sort of changes in somatic cells may ultimately be responsible for the series of degenerative processes occurring in most species after their reproductive period, generally termed aging or senescence. At first glance this possibility seems unlikely since there would be selection against it, for example, by the emergence of sophisticated antimutagenic systems, including D N A repair. However, from an evolutionary point of view, according to which aging is the result of late-acting deleterious genes--not selected against because of the declining force of natural selection with age (Charlesworth, 1980)---the emergence of systems for keeping the integrity of the somatic genome until eternity is unlikely (Kirkwood, 1977). Mutations are therefore likely to accumulate in the somatic genome of multicellular organisms. Indeed, rather than asking whether mutations accumulate, a more relevant question is to what extent somatic mutations contribute to cell degeneration and death and the increased incidence of disease that are so characteristic for the aging process. The concept of random genetic changes as a fundamental cause of senescence is still subject to controversy, in spite of the fact that some basic concepts of somatic mutation theories of aging have been formulated more than 30 years ago (Failla, 1958; Szilard, 1959). Probably the most elaborate theory is the somatic mutation theory of Burner (1974). In this theory it is postulated that senescence, which can be defined as the series of changes predisposing to fatal disease, is caused by the accumulation of D N A sequence changes as a consequence of imperfections of the enzymatic machinery for D N A replication, recombination and repair. Burnet ascribed a more important role to so-called intrinsic mutagenesis than to direct mutations via environmental agents. Consequently, he postulated that life span is determined by the intrinsic quality of the organism's D N A processing activities, e.g. D N A repair. Indeed, a direct correlation between species
lifespan and D N A repair rate has been postulated on the basis of excision repair measurements in cultured skin fibroblasts (Hart and Setlow, 1974). The complexity of the many different D N A repair pathways in higher animals, however, precludes any definite conclusion on the possible relationship between aging and D N A repair (Vijg et al., 1984; Vijg and Knook, 1987). In order to test the hypothesis that D N A mutations in the somatic cells of higher organisms are a major cause of senescence it is necessary to (1) quantitate and characterize D N A sequence changes in different organs and tissues as a function of age and (2) correlate these spectra of D N A defects to specific aging phenomena, such as age-related diseases. Studies to this aim are still in their infancy. Here we will briefly summarize the results obtained thus far in measuring different types of D N A sequence changes in vivo as a function of age. We will see that the interpretation of these results is often difficult because of limitations of the techniques used. Some new promising techniques for characterizing D N A sequence variation in different organs and tissues will be discussed. CHROMOSOMAL ABERRATIONS
An almost classical way of looking at D N A sequence changes is by scoring chromosomal aberrations (e.g. acentric fragments, dicentric chromosomes, centric ring chromosomes, reciprocal and non-reciprocal translocations, inversions) in metaphase plates. This can most easily be done by using cultured cells treated with colchicine, an inhibitor of spindle fiber formation. Chromosomal aberrations can then be scored microscopically by the inspection of metaphases. When the somatic mutation theory of aging was formulated for the first time there was no real possibility to experimentally test its predictions. In the beginning of the sixties Curtis and coworkers were the first to obtain some supportive evidence for the theory. In an extensive series of experiments, somatic mutations were studied as chromosomal aberrations
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in mouse liver cells, using partial hepatectomy to obtain metaphase plates of parenchymal cells (Curtis, 1966). By using this method they observed a steady increase in chromosomal aberrations with age. Indeed, while in 4-5-month-old mice about 10% of the cells had abnormal chromosomes, this percentage could become as high as 75% in mice older than 12 months (Stevenson and Curtis, 1961). It should be noted that the percentage of chromosome abnormalities at young age is already very high. Subsequent findings of Curtis and coworkers indicating a more rapid increase in chromosomal aberrations in short-lived as compared to long-lived mouse strains, and their observations that the life-shortening effect of radiation was accompanied by an increased level of chromosomal aberrations further strengthened the concept (Crowley and Curtis, 1963). The findings of Curtis and coworkers have been confirmed by many other authors for both humans and experimental animals. Martin et al. (1985), for example, reported a 5-fold higher frequency of chromosomal aberrations in primary cultures of kidney cells from 40-month-old mice as compared to young animals. The aberrations found most frequently were chromatid gaps and breaks, but also acentric fragments were observed. A number of studies have been performed on human peripheral blood lymphocytes. From the results of these studies the general consensus has emerged that the frequency of chromosomal aberrations greatly increases with age. Evans (1990) noted that in young non-cigarette smoking adults the frequency of ceils that are missing or have gained a chromosome (aneuploidy) is in the order of 1-2%. In another 1-2% of the cells there is breakage or other structural rearrangements. In 60-year-aids these frequencies may be six times higher. Indeed, the level of aneuploidy increases exponentially with age (Evans, 1990). Some large studies on chromosomal aberrations in humans in relation to age have been performed. Hedner et aL (1982) analyzed 200 peripheral blood lymphocytes from each of 100 individuals aged 18-65 years, and found an approximate 2-fold increase of the frequency of chromosomal aberrations with age. A more recent prospective study involved 1000 cells from each of only four young and two old subjects. The results of this study indicated a highly significant 3- and 9-fold age-related increase in chromosome breaks and gaps, respectively (Marlhens et al., 1986; Prieur et al., 1988). However, it should be noted that in both studies cells were in their second metaphase. It was suggested that many of the aberrations observed could have been induced by the culture conditions, for example, as a consequence of the switch these ceils undergo from a low to a high percentage of oxygen (H. Joenje, personal communication; see also Joenje, 1989). Esposito et al. (1989) provided evidence that cells of older individuals are more vulnerable to
the occurrence of chromosomal aberrations than younger persons. By using hybrids between Chinese hamster ovary cells deficient in HPRT (whose presence is essential for survival in HAT selective medium) and T-lymphocytes from young or old human individuals, these authors were able to screen for breakage along the human X chromosome, independent of cell survival. The results showed that the cell hybrid clones carrying an old-age X chromosome tended to lose an X chromosome short-arm marker during one month of culture more readily than those carrying a young-age X chromosome. Evidence was also provided that the old-age X chromosome is more sensitive to the clastogenic effects of aminopterine than a young-age X chromosome. Based on the pattern of marker loss in the old age-hybrid clones the authors ascribed spontaneous breakage to chromosomal regions prone to meiotic rearrangements and to constitutive and/or hereditary fragility. Such chromosomal fragile sites could function as hot spots of chromosomal aberrations during aging. Taken together, there is abundant evidence that the level of spontaneous gross D N A sequence abnormalities increases with age. The extent of this increase, however, is still subject to some controversy due to the limited number of studies and possible artefacts of the culture conditions. Indeed, in some studies no significant differences were seen in the frequency of chromosomal aberrations with respect to age (Anderson et al., 1988). Follow-up studies with many more individuals are therefore necessary both to confirm and extend the above described findings and to obtain a more complete picture of the types of aberrations that accumulate with age. Such studies could become feasible with the further sophistication of automated systems for scoring chromosomal aberrations, i.e. metaphase finders (Finnon et al., 1986) and flow-based methods (Carrano et aL, 1978; Green et al., 1989). SUBMICROSCOPICALDNA SEQUENCE CHANGES D N A rearrangements
D N A rearrangements occur as a normal, developmentally regulated process in a variety of organisms. In higher organisms the best example is the assemblage of genes coding for immunoglobulins and T-cell receptors during the development of the immune system (Hood et al., 1985). There is also evidence for the occurrence of D N A rearrangements in mouse brain during development (Matsuoka et al., 1991). Such processes of somatic recombination require breakage and rejoining of D N A segments. In protozoa breakage and rejoining form a mechanism for the elimination of D N A sequences during macronuclear development (Tausta and Klobutcher, 1990). Such processes are consistent; that is, they occur with a high frequency at certain defined positions in the genome of all cells in the population or species. The question arises whether such consistent
Somatic mutations and cellular aging rearrangements also occur during aging. Indeed, it is not inconceivable that the genome contains hotspots of genetic instability which could form the basis of genome degeneration during aging. For lower organisms it has been demonstrated that DNA rearrangements can form the basis of senescence. For example, in Podospora anserina senescence (replicative death after vegetative growth) is associated with the liberation from the mitochondrial genome of an intron in a gene which codes for an enzyme of the respiration chain (Esser, 1985). Once liberated from the mitochondria, the "mobile" intron circularizes and is subsequently able to propagate autonomously. More importantly, these mobile introns can be used to infect juvenile cells, which are then immediately transformed to senescence. Also in Neurospora DNA rearrangements are considered to be involved in senescence. In some rapidly senescing strains of this organism vegetative death seems to be caused by the insertion of DNA fragments into the mitochondrial chromosome (Bertrand et al., 1985; for a review on senescence in fungi, see Osiewacz, 1990). For higher organisms there is no evidence for consistent DNA rearrangements associated with senescence. However, it should be realized that such changes are extremely difficult to detect in view of the complexity of the genome of higher organisms. Nevertheless, attempts to demonstrate such changes have been made. Strehler was perhaps the first who pointed to repetitive sequences as likely candidates to undergo rearrangements that could be detected as gene copy loss during aging. Indeed, evidence was provided for a decrease in rRNA gene copy number in the genome of aging beagle dogs (Johnson and Strehler, 1972). As yet, however, these findings do not seem to have been confirmed or extended by others. Some moderately repeated sequences can be analyzed directly for any sign of instability in the genome. Indeed, work from Thein et al. (1987) indicated extensive changes in the sequence organization of minisatellites in human cancer DNA. Of 35 cases in which constitutional DNA was compared with tumor DNA alterations were observed, comprising both new length alleles and intensity changes of hybridizing DNA fragments. Comparable results were obtained by Armour et al. (1989), who used locus-specific probes and were therefore able to demonstrate that the mutation incidence in the tumors varied from locus to locus. In other words, in the genome of tumor cells there appear to be hotspots of rearrangements. That minisatellite repeats can act as hotspots of recombination was recently demonstrated by Wahls et al. (1990), who showed that a minisatellite consensus sequence stimulated homologous recombination up to 13.5-fold. When DNA rearrangements occur in tumors the question may arise whether they also occur in the
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tissue from which they originate, i.e. during development and aging. Kelly et al. (1989) showed that during mouse early development a hypermutable locus detected by a human minisatellite probe was subject to somatic mutation events resulting in mosaic mice. Data from Jeffreys et al. (1990) also indicate somatic mutations at a minisatellite locus in human blood DNA at a rate of 0.0006 per haploid genome. In a straightforward attempt to study DNA rearrangements in mammalian cells during aging, Slagboom et al. (1991) studied mini- and microsatellite sequences in fibroblast clones, derived from primary populations isolated from the skin of old and young rats. Using a novel genome scanning technique, 2-dimensional DNA typing (Uitterlinden et al., 1989), a total of about 3000 DNA fragments were analyzed for the occurrence of age-related fragment size polymorphisms. The data obtained indicated a mutation frequency as high as 4.5 x 10-3, confirming and extending (due to the large number of loci tested) the findings cited above. No differences were observed in relation to donor age. However, the number of different fibroblast clones analyzed was too small to draw any definite conclusions. In contrast to the DNA of the genome, mitochondrial DNA with its relatively small size of 16,569 bp lends itself well to studying rearrangements in mammals with age. Electron microscopic analysis of mouse mitochondrial DNA after denaturation/ renaturation indicated an increased level of deletion/insertion events (involving fragments of about 400 bp) in old as compared to young animals (Piko et al., 1988). Deletions in mitochondrial DNA have been shown to be present in patients affected by mitochondrial myopathies and encephalomyopathies. It has been suggested by Linnane and coworkers that low levels of such deletions would occur in all humans and accumulate in an age-related way (Linnane et al., 1992; Arnheim and Cortopassi, 1992). The potential role of mitochondrial DNA sequence changes in aging and disease of humans as compared to the situation in lower organisms like Podospora has recently been reviewed (Osiewacz, 1992). Gene mutations
DNA sequence changes in expressed genes can be measured indirectly by means of selection, using a wide variety of phenotypic characteristics. In this way it is possible to measure mutation rate at a given locus by determining the ratio of changed versus non-changed phenotype of a given character This principle is most readily applied with bacteria as has been demonstrated by the pioneering work of Ames and coworkers, who applied histidine-requiring strains of the bacterium Salmonella typhimurium to demonstrate mutagenesis. In this case one is not scoring forward, but back mutations, i.e. the reversion from histidine requirement to growth on
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minimal medium (Ames et al., 1973 and references therein). Ames et al. (1973) also described a forward mutagenesis test, which for genotoxicity purposes was considered less sensitive than a specific backmutation test in view of the many spontaneous mutants of various types. This of course is precisely the reason why in aging research forward mutation systems are desirable. An example of a forward mutation system that can be applied to detect spontaneous mutations in mammalian cells is the HPRT test. The gene for hypoxanthine guanine phosphoribosyl transferase (HPRT) is an X-linked gene that upon inactivation confers resistance to purine analogs, such as 6-thioguanine. Because of this X-linkage, only one allele needs to be mutated in both males (one X chromosome) and females (one active X chromosome). The HPRT assay can be applied on cells that are still capable of proliferating, such as cell lines of different sources, primary human fibroblasts and primary fibroblastoid and epitheliold cells from various organs and tissues of rodents. The most reliable results are obtained when the technique is applied in the form of a clonal assay; only then is it possible to confirm the mutant nature of the 6-thioguanine resistant cell. After culturing in the presence of 6-thioguanine the frequency of resistant clones can be considered as the mutant frequency. Characterization of the mutations is possible after D N A isolation from the expanded clones followed by standard hybridization analysis, using HPRT probes. The HPRT system is thus suitable for the detection of all classes of mutational events, provided they inactivate the gene. A distinct advantage offered by the HPRT method should be stressed. Since this endogenous gene is naturally present in human cells it is possible to measure the percentage of mutant T-cells in human peripheral blood lymphocytes (Albertini et al., 1982). Such cells, which can be stimulated to divide and subsequently cloned could serve as an index for somatic mutation frequency in vivo. However, it should be noted that the observed mutant frequencies may not always be representative for the true in vivo mutant frequencies (Featherstone et al., 1987). Another system that can also be applied on human cells in vivo is an antibody-based test for mutations at the HLA-A locus, which is part of the human major histocompatibility region on chromosome 6. By tagging cells bearing HLA-A2/A3 with specific antibodies against one of these gene products and subsequent elimination of the wild type cells by antibody-complement mediated cytotoxicity, cells mutated at HLA-A can be selected (Janatipour et al., 1988). This system can be applied to study/n vivo mutant frequencies in individuals heterozygous for either HLA-A2 or HLA-A3 (about half of all individuals). From numerous studies applying the HPRT system, it has become clear that the spontaneous mutation frequency at this locus is in the order of 10 -6
to 10 -5 and increases with age. This increase in mutant frequencies is highly significant and may be as high as one order of magnitude, from neonates to people over 60 (Evans and Vijayalaxmi, 1981; Morley et aL, 1982; Trainor et al., 1984; Carrano, 1989). However, the large inter-individual differences preclude any accurate estimate. The available data do also not allow us to deduce whether the age-related mutation accumulation is linear or exponential. Regarding the nature of the mutations detected in this system most are point mutations, but a considerable part are rearrangements. Analysis of the HPRT gene in human T-cells in vivo showed that among 30 HPRT deficient mutants obtained from seven normal control individuals six were deletions of up to 38 kb (Bradley et al. 1987). More or less comparable results were obtained for the HLA-A locus test. Using this system mutant frequencies as well as mutation spectra were determined as a function of donor age (Grist et al., 1992). Like in the HPRT test, most mutations in HLA-A were point mutations (about 65%). Interestingly, the HLA-A data indicate that approximately one-third of the mutants were due to mitotic recombination events that can not be scored in the HPRT test. The frequency of this type of mutations increased significantly with age, like the frequency of point mutations. From the data it is quite clear that inter-individual variation for these types of mutations strongly increases with age, which does not seem to be the case for deletion mutations. A general disadvantage of the use of endogenous selectable genes is that both the selection process and the characterization of the mutations is time-consuming. In addition, their application is usually limited to cells that can be stimulated to proliferate in vitro; direct detection of mutations in different organs and tissues is not possible. To some extent a solution to this latter problem was offered by the elegant procedure to detect somatic mutations at the glucose-6phosphate dehydrogenase locus in situ, by X-linked enzyme histochemistry (Griffiths et al., 1988) and by the use of mice heterozygous for the Dolichos biflorus agglutinin locus (Winton et aL, 1988; Schmidt et al., 1990). These methods, however, are still technically immature (see Nature 352, 200-201, scientific correspondence), can only be used in particular tissues and also suffer from the drawback that mutations can not be characterized. However, especially in situ, methods for demonstrating somatic mutations are invaluable since they allow us to determine mutation frequency at a specific locus in different cell types (expressing the mutational target gene) and in different areas of tissues and organs. In order to obtain more extensive information on mammalian mutagenesis the use of shuttle vectors has been advocated as a good alternative to the tedious procedures associated with selectable endogenous genes. Shuttle vectors provided with a marker gene can be introduced into mammalian
Somatic mutations and cellular aging
cells and then retrieved ("shuttled") to bacteria in which selection can take place between mutated and non-mutated marker genes. Since the marker gene is already cloned, characterization by nucleotide sequencing is now easy. The first results with extrachromosomal shuttle vectors, based on the SV40 origin of replication for autonomous replication in mammalian cells, were disappointing, due to the high background mutation frequencies observed (Razzaque et al., 1983; Calos et al., 1983). This problem, which should probably be ascribed to damage inflicted on the incoming vector DNA by cellular enzymes during transfection, has been solved to a certain extent by using other vectors (DuBridge et al., 1987). Nevertheless, extrachromosomal vectors will always have the major disadvantage that they do not resemble the natural environment of mammalian endogenous genes. This is in contrast to integrated shuttle vectors, which can be considered as more representative for genomic susceptibility to mutagenesis than extrachromosomal vectors. Also the background mutation frequency of integrated vectors should be lower, since cells with only intact copies of the vector can be selected. Integrated shuttle vectors provided with supF mutational target genes have been used, but their rescue appeared to be highly inefficient (Glazer et al., 1986). Gossen and Vijg (1988), provided evidence that integrated bacteriophage lambda shuttle vectors in chromosomal DNA of transgenic mice are highly methylated. They suggested that this could lead to host restriction during in vitro packaging or after retrieval to bacteria. By using the host restriction negative strain E.coli C, high rescue efficiencies were obtained (Gossen and Vijg, 1988; Gossen et al., 1989). To apply the shuttle vector principle for mutation detection in vivo Gossen et al. (1989) generated transgenic mouse strains harboring multiple copies of tandemly integrated bacteriophage lambda shuttle vectors. The system has the advantage of low background mutation frequency and offers the possibility of detecting and characterizing mutations in various organs and tissues (Gossen et aL, 1989; Gossen and Vijg, 1990). By using this model it should be possible to study spontaneous mutation frequencies and the relationship of DNA damage and repair to mutagenesis, carcinogenesis and aging in different organs and tissues (Vijg et al., 1990). Thus far some limited datasets have been obtained on spontaneous and induced mutations in different organs and tissues of these animals. Spontaneous mutation frequencies appeared to be in the same order as had been found earlier for the HPRT and HLA locus, i.e. about 10 -5 (Gossen et al., unpublished). In general, tissues with a high mitotic activity like bone marrow appear to have a relatively high mutation frequency as compared to postmitotic tissues like the brain. This is the case for at least four vector-containing transgenic animal strains. How-
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ever, one important exception has emerged thus far. In a particular strain with the bacteriophage lambda cluster near the pseudoautosomal region on the X-chromosome spontaneous mutation frequencies appeared to be 10-100 times higher (Gossen et al., 1991). Apparently, spontaneous mutation frequency depends on the chromosomal position. Sequence analysis of the mutants obtained revealed point mutations as the predominant type of spontaneous mutations. Especially GC to AT transitions at CpG sites were frequently found, indicating deaminations of 5-methylcytosine. This is in keeping with observation of CpG methylation in normal human tissues at sites accounting for more than 30% of germ-line mutations in the low-density lipoprotein receptor gene or somatic mutations in the p53 tumor suppressor gene (Rideout Ill et al., 1990). It is also in agreement with the high percentage of point mutations observed in HPRT and HLA-A genes (Grist et al., 1992). Like in the HPRT and HLA-A genes, deletion mutations form a minor fraction in the mutation spectra observed. In this respect it should be noted that with the presently available bacteriophage lambda-based transgenic mouse models scoring of deletions larger than about 10 kb is not possible. Recently some important modifications of the mutation detection step in these systems has greatly reduced the time and effort to carry out large-scale mutation studies. Indeed, the construction of Gal E bacterial hosts facilitate the positive selection of mutants, thereby eliminating the necessity to screen large numbers of plaques (or colonies with plasmidbased systems) to find the few mutants (Gossen et al., 1992, 1993). In Fig. 1 the different transgenie mutation models are schematically depicted (reviewed by Gossen and Vijg, 1993a,b). Thus, the newly emerged transgenic mouse models appear to be valid models for studying mutagenesis in different organs and tissues. Indeed, the relative ease with which in these systems mutant frequencies can accurately be determined and large numbers of mutants can be characterized allows for the first time to test directly some basic aspects of the somatic mutation theory of aging. As yet no results with respect to mutant frequencies and mutation spectra with age have been reported. Such data, however, are expected soon and will finally provide the long desired accurate data on type and frequencies of mutations in different organs and tissues in relation to age. The development of transgenic mutation models is a major step forward in the path toward the ultimate method for testing somatic mutation theories of aging. Nevertheless, it should be realized that in all cases the mutational target is only a very small part of the genome and, even with integrated genes, not necessarily representative. The ideal way of determining the type and frequency of gene mutations in somatic cells in relation to aging would be by direct
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+/- Chemical treatment Collect tissues
Genomic D N A isolation i ~ / In vitro packag
Restriction ~ enzyme digestion ~ Circularization " ~ b y ligation
Lambda rescue
Plasmid rescue
Q LacZ-based
/
@© Rescue efficiency
@©
Mutants colourless
Mutants blue and non-mutants colourless
Mutation frequency =
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# Mutant reporter genes Total # reporter genes analyzed
Fig. 1. Overview of transgenic mouse mutation models (from Gossen and Vijg, 1992). analysis, using the large reservoir of DNA probes that is presently available. This approach requires a methodology that is powerful enough to first separate one mutant gene from approximately 100,000 mutant genes and subsequently quantitate and characterize the mutant. A procedure based on the use of denaturing gradient gel electrophoresis to separate PCRamplified target sequences in one major wild type band and a number of early melting heteroduplex mutant molecules has been designed by Vijg and Uitterlinden (1987). Thilly and coworkers demonstrated that it is possible to separate in denaturing gradient gels mutant HPRT sequences obtained from uncloned, complex populations of human B-cells treated with different mutagens and cultured in the presence of 6-thioguanine (Thilly et al., 1989). However, this is still far away from detecting mutants without applying any selection. Indeed, the crucial step in these protocols is the PCR amplification step, the error rate of which should be sufficiently low not to overshadow the mutations that should be detected. In a comparative
study it was found that the PCR error rate could be greatly reduced by using high-fidelity polymerases, such as T4 and T7, and by alterations of the experimental conditions (Keohavong and Thilly, 1989). Cariello et al. (1991) examined the fidelity of several DNA polymerases used in PCR and found Vent polymerase to cause the least amount of errors, i.e. 2.4 × l0 -5. This is still too high, however, to make direct mutation analysis without selection a feasible strategy. An interesting method was developed by Sandy et al. 0992). In this system, point mutations in Taq I restriction sites are detected by the selective amplification of mutated recognition sequences; the mutant fragments rescued in this way are directly sequenced or identified by oligonucleotide hybridization. Because of the extremely small mutational target the risk of PCR-related errors is small, although some were observed (Sandy et al., 1992). However, this very small mutational target is also the method's greatest disadvantage; although it allows the measurement of spontaneous mutations in human
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Somatic mutations and cellular aging and animal tissues without selection, it may not be very representative for the total of mutational events occurring with age. SUMMARY AND GENERAL DISCUSSION
The major conclusion that can be drawn from this overview on somatic mutations and cellular aging is that the increased mutation load predicted from the inherent imperfection of DNA maintenance and repair systems seems to be present. For chromosomal aberrations the majority of reports indicate increased levels with age. The same is true for mutations present in HPRT and HLA-A in human blood cells. This, however, does not give us a clear picture of the mutation load in different organs and tissues. Neither does it provide information about the kinetics of mutation accumulation. The accumulation of mutations in a linear fashion is not likely to lead to extremely high levels at old age and would therefore suggest mutation accumulation to be a consequence rather than a cause of aging. However, an exponential increase, for example, due to a decrease in the proficiency of cellular defense (e.g. DNA repair, anti-oxidant systems), could lead to extremely high levels of mutations at old age. The very slow emergence of hard data on mutation accumulation with age has been accompanied, especially during the last couple of years, by the development of methods that allow mutation detection and characterization at a large scale. In the context of ongoing studies toward that aim, it could be useful to speculate about how mutations contribute to aging. In this regard one can ask the following question: at which frequency and at which loci should mutations occur to cause the specific age-related degenerations that can be observed? The answer clearly should take into account the possibility that mutant somatic cells can be lost from the population, either as a consequence of the mutation itself or by programmed cell death as a sort of self-protection against too high a mutation load. Selection is a good explanation for the apparent immortality of the germ-line; even after conception approximately 15-20% of pregnancies terminate in spontaneous abortion as a consequence of chromosomal anomalies (Plachot et al., 1987; Minguillon et al., 1989; see, also, Vijg, 1990). Although such selection leading to cell death will certainly occur after the occurrence of mutational events in crucial (housekeeping) genes, the question remains as to what would happen when mutational events only result in slightly changed gene expression levels. In cell cultures this would most likely lead to growth (dis)advantages of particular cells and hence the retention of uniformity. In multicellular organisms, however, the static distribution of cells over a great variety of organs and tissues would result in a mosaic, much like the situation in certain single plants (Sutherland and Watkinson, 1986). The question then
remains as to how much genetic variability would be necessary to explain age-related functional decline and death. Two lines of research should, within several years, provide the answers. First, accurate data on mutation loads in different organs and tissues of the recently generated transgenic mouse models can be expected soon. Such data can then be correlated with patterns of age-related multiple pathology and particular forms of age-related functional decline. Second, mathematical modeling using the wealth of information on the regulatory control of many genes in the human and animal genomes that are presently generated, may provide some answers to the question of how much mutations and in what sequences are necessary to cause particular agerelated defects. REFERENCES
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Somatic mutations and cellular aging Morley A., Cox S. and Holliday R. (1982) Human lymphocytes resistant to 6-thiognanine increase with age. Mech. Age. Devl. 19, 21-26. Mullaart E., Verwest A., Morolli B., te Meetman G. J., Uitterlinden, A. G. and Vijg J. (1992) Rapid genome scanning by two-dimensional DNA typing: applications in the analysis of getically complex traits. Submitted for publication. Osiewacz H. D. (1990) Molecular analysis of aging processes in fungi. Murat. Res. 237, 1-8. Osiewacz, H. D. and Hermanns, J. (1992) The role of mitochondrial DNA in aging and human diseases. Aging, 4, 273-286. Piko L., Hougham A. J. and Bullpit K. J. (1988) Studies of sequence heterogeneity of mitochondrial DNA from rat and mouse tissues: evidence for an increased frequency of deletions/additions with aging. Mech. Ageing Devl. 43, 279-293. Plachot M., De Grouchi J., Junca A-M., Mandelbaum J., Turleau C., Couillin P., Cohen J. and Salat-Baroux J. (1987) From oocyte to embryo: a model, deduced from in vitro fertilization, for natural selection against chromosome abnormalities. Ann. G~n~t. 30, 22-32. Prieur M., Achkar, W. A. I., Aurias A., Couturier J., Dutrillaux A. M., Flury-Herard A., Gerbault-Seureau M., Hoffschir F., Lamoliatte E., Lefrancois D., Lombard M., Muleris M., Ricoul M., Sabatier L. and ViegasPequignot E. (1988) Aquired chromosome rearrangements in human lymphocytes: effects of aging. Hum. Genet. 79, 147-150. Razzaque A., Mizusawa H., Seidman M. M. (1983) Rearrangement and mutagenesis of a shuttle vector plasmid after passage in mammalian cells. Proc. Nam. Acad. Sci. U.S.A. 80, 3010-3014. Rideout III W. M., Coetzee G. A., Olumi A. F. and Jones P. A. (1990) 5-Methylcytosine as an endogenous mutagen in the human LDL receptor and p53 genes. Science 249, 1288-1290. Sandy M. S., Chiocca S. M. and Cerutti P. A. (1992) Genotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction. Proc. Nam. Acad. Sci. U.S.A. 89, 890-894. Schmidt G. H., O'Sullivan J. and Paul D. (1990) Ethylnitrosourea-induced mutations in vivo involving the Dolichos biflorus agglutinin receptor in mouse intestinal epithelium. Mutat. Res. 228, 149-155. Slagboom P. E., Mullaart E., Droog S. and Vijg J. (1991) Somatic mutations and cellular aging: two-dimensional DNA typing of rat fibroblast clones. Mutat. Res. 256, 311-321. Stevenson K. G. and Curtis H. J. (1961) Chromosomal
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M I N I REVIEW
SOMATIC MUTATIONS AND CELLULAR AGING J. VIJG and J. A. GossEr~ Harvard Medical School, Division on Aging, 643 Huntington Avenue, Boston, MA 02115, U.S.A.; and Ingeny B.V., P.O. Box 685, 2300 Ar Leiden, The Netherlands (Fax 31 71 210236) (Received 7 October 1992; accepted I 1 November 1992)
Changes in the D N A information content are fundamental to the continuous emergence of new life forms through evolution by natural selection. In multicellular organisms, however, the same sort of changes in somatic cells may ultimately be responsible for the series of degenerative processes occurring in most species after their reproductive period, generally termed aging or senescence. At first glance this possibility seems unlikely since there would be selection against it, for example, by the emergence of sophisticated antimutagenic systems, including D N A repair. However, from an evolutionary point of view, according to which aging is the result of late-acting deleterious genes--not selected against because of the declining force of natural selection with age (Charlesworth, 1980)---the emergence of systems for keeping the integrity of the somatic genome until eternity is unlikely (Kirkwood, 1977). Mutations are therefore likely to accumulate in the somatic genome of multicellular organisms. Indeed, rather than asking whether mutations accumulate, a more relevant question is to what extent somatic mutations contribute to cell degeneration and death and the increased incidence of disease that are so characteristic for the aging process. The concept of random genetic changes as a fundamental cause of senescence is still subject to controversy, in spite of the fact that some basic concepts of somatic mutation theories of aging have been formulated more than 30 years ago (Failla, 1958; Szilard, 1959). Probably the most elaborate theory is the somatic mutation theory of Burner (1974). In this theory it is postulated that senescence, which can be defined as the series of changes predisposing to fatal disease, is caused by the accumulation of D N A sequence changes as a consequence of imperfections of the enzymatic machinery for D N A replication, recombination and repair. Burnet ascribed a more important role to so-called intrinsic mutagenesis than to direct mutations via environmental agents. Consequently, he postulated that life span is determined by the intrinsic quality of the organism's D N A processing activities, e.g. D N A repair. Indeed, a direct correlation between species
lifespan and D N A repair rate has been postulated on the basis of excision repair measurements in cultured skin fibroblasts (Hart and Setlow, 1974). The complexity of the many different D N A repair pathways in higher animals, however, precludes any definite conclusion on the possible relationship between aging and D N A repair (Vijg et al., 1984; Vijg and Knook, 1987). In order to test the hypothesis that D N A mutations in the somatic cells of higher organisms are a major cause of senescence it is necessary to (1) quantitate and characterize D N A sequence changes in different organs and tissues as a function of age and (2) correlate these spectra of D N A defects to specific aging phenomena, such as age-related diseases. Studies to this aim are still in their infancy. Here we will briefly summarize the results obtained thus far in measuring different types of D N A sequence changes in vivo as a function of age. We will see that the interpretation of these results is often difficult because of limitations of the techniques used. Some new promising techniques for characterizing D N A sequence variation in different organs and tissues will be discussed. CHROMOSOMAL ABERRATIONS
An almost classical way of looking at D N A sequence changes is by scoring chromosomal aberrations (e.g. acentric fragments, dicentric chromosomes, centric ring chromosomes, reciprocal and non-reciprocal translocations, inversions) in metaphase plates. This can most easily be done by using cultured cells treated with colchicine, an inhibitor of spindle fiber formation. Chromosomal aberrations can then be scored microscopically by the inspection of metaphases. When the somatic mutation theory of aging was formulated for the first time there was no real possibility to experimentally test its predictions. In the beginning of the sixties Curtis and coworkers were the first to obtain some supportive evidence for the theory. In an extensive series of experiments, somatic mutations were studied as chromosomal aberrations
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in mouse liver cells, using partial hepatectomy to obtain metaphase plates of parenchymal cells (Curtis, 1966). By using this method they observed a steady increase in chromosomal aberrations with age. Indeed, while in 4-5-month-old mice about 10% of the cells had abnormal chromosomes, this percentage could become as high as 75% in mice older than 12 months (Stevenson and Curtis, 1961). It should be noted that the percentage of chromosome abnormalities at young age is already very high. Subsequent findings of Curtis and coworkers indicating a more rapid increase in chromosomal aberrations in short-lived as compared to long-lived mouse strains, and their observations that the life-shortening effect of radiation was accompanied by an increased level of chromosomal aberrations further strengthened the concept (Crowley and Curtis, 1963). The findings of Curtis and coworkers have been confirmed by many other authors for both humans and experimental animals. Martin et al. (1985), for example, reported a 5-fold higher frequency of chromosomal aberrations in primary cultures of kidney cells from 40-month-old mice as compared to young animals. The aberrations found most frequently were chromatid gaps and breaks, but also acentric fragments were observed. A number of studies have been performed on human peripheral blood lymphocytes. From the results of these studies the general consensus has emerged that the frequency of chromosomal aberrations greatly increases with age. Evans (1990) noted that in young non-cigarette smoking adults the frequency of ceils that are missing or have gained a chromosome (aneuploidy) is in the order of 1-2%. In another 1-2% of the cells there is breakage or other structural rearrangements. In 60-year-aids these frequencies may be six times higher. Indeed, the level of aneuploidy increases exponentially with age (Evans, 1990). Some large studies on chromosomal aberrations in humans in relation to age have been performed. Hedner et aL (1982) analyzed 200 peripheral blood lymphocytes from each of 100 individuals aged 18-65 years, and found an approximate 2-fold increase of the frequency of chromosomal aberrations with age. A more recent prospective study involved 1000 cells from each of only four young and two old subjects. The results of this study indicated a highly significant 3- and 9-fold age-related increase in chromosome breaks and gaps, respectively (Marlhens et al., 1986; Prieur et al., 1988). However, it should be noted that in both studies cells were in their second metaphase. It was suggested that many of the aberrations observed could have been induced by the culture conditions, for example, as a consequence of the switch these ceils undergo from a low to a high percentage of oxygen (H. Joenje, personal communication; see also Joenje, 1989). Esposito et al. (1989) provided evidence that cells of older individuals are more vulnerable to
the occurrence of chromosomal aberrations than younger persons. By using hybrids between Chinese hamster ovary cells deficient in HPRT (whose presence is essential for survival in HAT selective medium) and T-lymphocytes from young or old human individuals, these authors were able to screen for breakage along the human X chromosome, independent of cell survival. The results showed that the cell hybrid clones carrying an old-age X chromosome tended to lose an X chromosome short-arm marker during one month of culture more readily than those carrying a young-age X chromosome. Evidence was also provided that the old-age X chromosome is more sensitive to the clastogenic effects of aminopterine than a young-age X chromosome. Based on the pattern of marker loss in the old age-hybrid clones the authors ascribed spontaneous breakage to chromosomal regions prone to meiotic rearrangements and to constitutive and/or hereditary fragility. Such chromosomal fragile sites could function as hot spots of chromosomal aberrations during aging. Taken together, there is abundant evidence that the level of spontaneous gross D N A sequence abnormalities increases with age. The extent of this increase, however, is still subject to some controversy due to the limited number of studies and possible artefacts of the culture conditions. Indeed, in some studies no significant differences were seen in the frequency of chromosomal aberrations with respect to age (Anderson et al., 1988). Follow-up studies with many more individuals are therefore necessary both to confirm and extend the above described findings and to obtain a more complete picture of the types of aberrations that accumulate with age. Such studies could become feasible with the further sophistication of automated systems for scoring chromosomal aberrations, i.e. metaphase finders (Finnon et al., 1986) and flow-based methods (Carrano et aL, 1978; Green et al., 1989). SUBMICROSCOPICALDNA SEQUENCE CHANGES D N A rearrangements
D N A rearrangements occur as a normal, developmentally regulated process in a variety of organisms. In higher organisms the best example is the assemblage of genes coding for immunoglobulins and T-cell receptors during the development of the immune system (Hood et al., 1985). There is also evidence for the occurrence of D N A rearrangements in mouse brain during development (Matsuoka et al., 1991). Such processes of somatic recombination require breakage and rejoining of D N A segments. In protozoa breakage and rejoining form a mechanism for the elimination of D N A sequences during macronuclear development (Tausta and Klobutcher, 1990). Such processes are consistent; that is, they occur with a high frequency at certain defined positions in the genome of all cells in the population or species. The question arises whether such consistent
Somatic mutations and cellular aging rearrangements also occur during aging. Indeed, it is not inconceivable that the genome contains hotspots of genetic instability which could form the basis of genome degeneration during aging. For lower organisms it has been demonstrated that DNA rearrangements can form the basis of senescence. For example, in Podospora anserina senescence (replicative death after vegetative growth) is associated with the liberation from the mitochondrial genome of an intron in a gene which codes for an enzyme of the respiration chain (Esser, 1985). Once liberated from the mitochondria, the "mobile" intron circularizes and is subsequently able to propagate autonomously. More importantly, these mobile introns can be used to infect juvenile cells, which are then immediately transformed to senescence. Also in Neurospora DNA rearrangements are considered to be involved in senescence. In some rapidly senescing strains of this organism vegetative death seems to be caused by the insertion of DNA fragments into the mitochondrial chromosome (Bertrand et al., 1985; for a review on senescence in fungi, see Osiewacz, 1990). For higher organisms there is no evidence for consistent DNA rearrangements associated with senescence. However, it should be realized that such changes are extremely difficult to detect in view of the complexity of the genome of higher organisms. Nevertheless, attempts to demonstrate such changes have been made. Strehler was perhaps the first who pointed to repetitive sequences as likely candidates to undergo rearrangements that could be detected as gene copy loss during aging. Indeed, evidence was provided for a decrease in rRNA gene copy number in the genome of aging beagle dogs (Johnson and Strehler, 1972). As yet, however, these findings do not seem to have been confirmed or extended by others. Some moderately repeated sequences can be analyzed directly for any sign of instability in the genome. Indeed, work from Thein et al. (1987) indicated extensive changes in the sequence organization of minisatellites in human cancer DNA. Of 35 cases in which constitutional DNA was compared with tumor DNA alterations were observed, comprising both new length alleles and intensity changes of hybridizing DNA fragments. Comparable results were obtained by Armour et al. (1989), who used locus-specific probes and were therefore able to demonstrate that the mutation incidence in the tumors varied from locus to locus. In other words, in the genome of tumor cells there appear to be hotspots of rearrangements. That minisatellite repeats can act as hotspots of recombination was recently demonstrated by Wahls et al. (1990), who showed that a minisatellite consensus sequence stimulated homologous recombination up to 13.5-fold. When DNA rearrangements occur in tumors the question may arise whether they also occur in the
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tissue from which they originate, i.e. during development and aging. Kelly et al. (1989) showed that during mouse early development a hypermutable locus detected by a human minisatellite probe was subject to somatic mutation events resulting in mosaic mice. Data from Jeffreys et al. (1990) also indicate somatic mutations at a minisatellite locus in human blood DNA at a rate of 0.0006 per haploid genome. In a straightforward attempt to study DNA rearrangements in mammalian cells during aging, Slagboom et al. (1991) studied mini- and microsatellite sequences in fibroblast clones, derived from primary populations isolated from the skin of old and young rats. Using a novel genome scanning technique, 2-dimensional DNA typing (Uitterlinden et al., 1989), a total of about 3000 DNA fragments were analyzed for the occurrence of age-related fragment size polymorphisms. The data obtained indicated a mutation frequency as high as 4.5 x 10-3, confirming and extending (due to the large number of loci tested) the findings cited above. No differences were observed in relation to donor age. However, the number of different fibroblast clones analyzed was too small to draw any definite conclusions. In contrast to the DNA of the genome, mitochondrial DNA with its relatively small size of 16,569 bp lends itself well to studying rearrangements in mammals with age. Electron microscopic analysis of mouse mitochondrial DNA after denaturation/ renaturation indicated an increased level of deletion/insertion events (involving fragments of about 400 bp) in old as compared to young animals (Piko et al., 1988). Deletions in mitochondrial DNA have been shown to be present in patients affected by mitochondrial myopathies and encephalomyopathies. It has been suggested by Linnane and coworkers that low levels of such deletions would occur in all humans and accumulate in an age-related way (Linnane et al., 1992; Arnheim and Cortopassi, 1992). The potential role of mitochondrial DNA sequence changes in aging and disease of humans as compared to the situation in lower organisms like Podospora has recently been reviewed (Osiewacz, 1992). Gene mutations
DNA sequence changes in expressed genes can be measured indirectly by means of selection, using a wide variety of phenotypic characteristics. In this way it is possible to measure mutation rate at a given locus by determining the ratio of changed versus non-changed phenotype of a given character This principle is most readily applied with bacteria as has been demonstrated by the pioneering work of Ames and coworkers, who applied histidine-requiring strains of the bacterium Salmonella typhimurium to demonstrate mutagenesis. In this case one is not scoring forward, but back mutations, i.e. the reversion from histidine requirement to growth on
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minimal medium (Ames et al., 1973 and references therein). Ames et al. (1973) also described a forward mutagenesis test, which for genotoxicity purposes was considered less sensitive than a specific backmutation test in view of the many spontaneous mutants of various types. This of course is precisely the reason why in aging research forward mutation systems are desirable. An example of a forward mutation system that can be applied to detect spontaneous mutations in mammalian cells is the HPRT test. The gene for hypoxanthine guanine phosphoribosyl transferase (HPRT) is an X-linked gene that upon inactivation confers resistance to purine analogs, such as 6-thioguanine. Because of this X-linkage, only one allele needs to be mutated in both males (one X chromosome) and females (one active X chromosome). The HPRT assay can be applied on cells that are still capable of proliferating, such as cell lines of different sources, primary human fibroblasts and primary fibroblastoid and epitheliold cells from various organs and tissues of rodents. The most reliable results are obtained when the technique is applied in the form of a clonal assay; only then is it possible to confirm the mutant nature of the 6-thioguanine resistant cell. After culturing in the presence of 6-thioguanine the frequency of resistant clones can be considered as the mutant frequency. Characterization of the mutations is possible after D N A isolation from the expanded clones followed by standard hybridization analysis, using HPRT probes. The HPRT system is thus suitable for the detection of all classes of mutational events, provided they inactivate the gene. A distinct advantage offered by the HPRT method should be stressed. Since this endogenous gene is naturally present in human cells it is possible to measure the percentage of mutant T-cells in human peripheral blood lymphocytes (Albertini et al., 1982). Such cells, which can be stimulated to divide and subsequently cloned could serve as an index for somatic mutation frequency in vivo. However, it should be noted that the observed mutant frequencies may not always be representative for the true in vivo mutant frequencies (Featherstone et al., 1987). Another system that can also be applied on human cells in vivo is an antibody-based test for mutations at the HLA-A locus, which is part of the human major histocompatibility region on chromosome 6. By tagging cells bearing HLA-A2/A3 with specific antibodies against one of these gene products and subsequent elimination of the wild type cells by antibody-complement mediated cytotoxicity, cells mutated at HLA-A can be selected (Janatipour et al., 1988). This system can be applied to study/n vivo mutant frequencies in individuals heterozygous for either HLA-A2 or HLA-A3 (about half of all individuals). From numerous studies applying the HPRT system, it has become clear that the spontaneous mutation frequency at this locus is in the order of 10 -6
to 10 -5 and increases with age. This increase in mutant frequencies is highly significant and may be as high as one order of magnitude, from neonates to people over 60 (Evans and Vijayalaxmi, 1981; Morley et aL, 1982; Trainor et al., 1984; Carrano, 1989). However, the large inter-individual differences preclude any accurate estimate. The available data do also not allow us to deduce whether the age-related mutation accumulation is linear or exponential. Regarding the nature of the mutations detected in this system most are point mutations, but a considerable part are rearrangements. Analysis of the HPRT gene in human T-cells in vivo showed that among 30 HPRT deficient mutants obtained from seven normal control individuals six were deletions of up to 38 kb (Bradley et al. 1987). More or less comparable results were obtained for the HLA-A locus test. Using this system mutant frequencies as well as mutation spectra were determined as a function of donor age (Grist et al., 1992). Like in the HPRT test, most mutations in HLA-A were point mutations (about 65%). Interestingly, the HLA-A data indicate that approximately one-third of the mutants were due to mitotic recombination events that can not be scored in the HPRT test. The frequency of this type of mutations increased significantly with age, like the frequency of point mutations. From the data it is quite clear that inter-individual variation for these types of mutations strongly increases with age, which does not seem to be the case for deletion mutations. A general disadvantage of the use of endogenous selectable genes is that both the selection process and the characterization of the mutations is time-consuming. In addition, their application is usually limited to cells that can be stimulated to proliferate in vitro; direct detection of mutations in different organs and tissues is not possible. To some extent a solution to this latter problem was offered by the elegant procedure to detect somatic mutations at the glucose-6phosphate dehydrogenase locus in situ, by X-linked enzyme histochemistry (Griffiths et al., 1988) and by the use of mice heterozygous for the Dolichos biflorus agglutinin locus (Winton et aL, 1988; Schmidt et al., 1990). These methods, however, are still technically immature (see Nature 352, 200-201, scientific correspondence), can only be used in particular tissues and also suffer from the drawback that mutations can not be characterized. However, especially in situ, methods for demonstrating somatic mutations are invaluable since they allow us to determine mutation frequency at a specific locus in different cell types (expressing the mutational target gene) and in different areas of tissues and organs. In order to obtain more extensive information on mammalian mutagenesis the use of shuttle vectors has been advocated as a good alternative to the tedious procedures associated with selectable endogenous genes. Shuttle vectors provided with a marker gene can be introduced into mammalian
Somatic mutations and cellular aging
cells and then retrieved ("shuttled") to bacteria in which selection can take place between mutated and non-mutated marker genes. Since the marker gene is already cloned, characterization by nucleotide sequencing is now easy. The first results with extrachromosomal shuttle vectors, based on the SV40 origin of replication for autonomous replication in mammalian cells, were disappointing, due to the high background mutation frequencies observed (Razzaque et al., 1983; Calos et al., 1983). This problem, which should probably be ascribed to damage inflicted on the incoming vector DNA by cellular enzymes during transfection, has been solved to a certain extent by using other vectors (DuBridge et al., 1987). Nevertheless, extrachromosomal vectors will always have the major disadvantage that they do not resemble the natural environment of mammalian endogenous genes. This is in contrast to integrated shuttle vectors, which can be considered as more representative for genomic susceptibility to mutagenesis than extrachromosomal vectors. Also the background mutation frequency of integrated vectors should be lower, since cells with only intact copies of the vector can be selected. Integrated shuttle vectors provided with supF mutational target genes have been used, but their rescue appeared to be highly inefficient (Glazer et al., 1986). Gossen and Vijg (1988), provided evidence that integrated bacteriophage lambda shuttle vectors in chromosomal DNA of transgenic mice are highly methylated. They suggested that this could lead to host restriction during in vitro packaging or after retrieval to bacteria. By using the host restriction negative strain E.coli C, high rescue efficiencies were obtained (Gossen and Vijg, 1988; Gossen et al., 1989). To apply the shuttle vector principle for mutation detection in vivo Gossen et al. (1989) generated transgenic mouse strains harboring multiple copies of tandemly integrated bacteriophage lambda shuttle vectors. The system has the advantage of low background mutation frequency and offers the possibility of detecting and characterizing mutations in various organs and tissues (Gossen et aL, 1989; Gossen and Vijg, 1990). By using this model it should be possible to study spontaneous mutation frequencies and the relationship of DNA damage and repair to mutagenesis, carcinogenesis and aging in different organs and tissues (Vijg et al., 1990). Thus far some limited datasets have been obtained on spontaneous and induced mutations in different organs and tissues of these animals. Spontaneous mutation frequencies appeared to be in the same order as had been found earlier for the HPRT and HLA locus, i.e. about 10 -5 (Gossen et al., unpublished). In general, tissues with a high mitotic activity like bone marrow appear to have a relatively high mutation frequency as compared to postmitotic tissues like the brain. This is the case for at least four vector-containing transgenic animal strains. How-
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ever, one important exception has emerged thus far. In a particular strain with the bacteriophage lambda cluster near the pseudoautosomal region on the X-chromosome spontaneous mutation frequencies appeared to be 10-100 times higher (Gossen et al., 1991). Apparently, spontaneous mutation frequency depends on the chromosomal position. Sequence analysis of the mutants obtained revealed point mutations as the predominant type of spontaneous mutations. Especially GC to AT transitions at CpG sites were frequently found, indicating deaminations of 5-methylcytosine. This is in keeping with observation of CpG methylation in normal human tissues at sites accounting for more than 30% of germ-line mutations in the low-density lipoprotein receptor gene or somatic mutations in the p53 tumor suppressor gene (Rideout Ill et al., 1990). It is also in agreement with the high percentage of point mutations observed in HPRT and HLA-A genes (Grist et al., 1992). Like in the HPRT and HLA-A genes, deletion mutations form a minor fraction in the mutation spectra observed. In this respect it should be noted that with the presently available bacteriophage lambda-based transgenic mouse models scoring of deletions larger than about 10 kb is not possible. Recently some important modifications of the mutation detection step in these systems has greatly reduced the time and effort to carry out large-scale mutation studies. Indeed, the construction of Gal E bacterial hosts facilitate the positive selection of mutants, thereby eliminating the necessity to screen large numbers of plaques (or colonies with plasmidbased systems) to find the few mutants (Gossen et al., 1992, 1993). In Fig. 1 the different transgenie mutation models are schematically depicted (reviewed by Gossen and Vijg, 1993a,b). Thus, the newly emerged transgenic mouse models appear to be valid models for studying mutagenesis in different organs and tissues. Indeed, the relative ease with which in these systems mutant frequencies can accurately be determined and large numbers of mutants can be characterized allows for the first time to test directly some basic aspects of the somatic mutation theory of aging. As yet no results with respect to mutant frequencies and mutation spectra with age have been reported. Such data, however, are expected soon and will finally provide the long desired accurate data on type and frequencies of mutations in different organs and tissues in relation to age. The development of transgenic mutation models is a major step forward in the path toward the ultimate method for testing somatic mutation theories of aging. Nevertheless, it should be realized that in all cases the mutational target is only a very small part of the genome and, even with integrated genes, not necessarily representative. The ideal way of determining the type and frequency of gene mutations in somatic cells in relation to aging would be by direct
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+/- Chemical treatment Collect tissues
Genomic D N A isolation i ~ / In vitro packag
Restriction ~ enzyme digestion ~ Circularization " ~ b y ligation
Lambda rescue
Plasmid rescue
Q LacZ-based
/
@© Rescue efficiency
@©
Mutants colourless
Mutants blue and non-mutants colourless
Mutation frequency =
Rescue efficiency
Mutants colourless
# Mutant reporter genes Total # reporter genes analyzed
Fig. 1. Overview of transgenic mouse mutation models (from Gossen and Vijg, 1992). analysis, using the large reservoir of DNA probes that is presently available. This approach requires a methodology that is powerful enough to first separate one mutant gene from approximately 100,000 mutant genes and subsequently quantitate and characterize the mutant. A procedure based on the use of denaturing gradient gel electrophoresis to separate PCRamplified target sequences in one major wild type band and a number of early melting heteroduplex mutant molecules has been designed by Vijg and Uitterlinden (1987). Thilly and coworkers demonstrated that it is possible to separate in denaturing gradient gels mutant HPRT sequences obtained from uncloned, complex populations of human B-cells treated with different mutagens and cultured in the presence of 6-thioguanine (Thilly et al., 1989). However, this is still far away from detecting mutants without applying any selection. Indeed, the crucial step in these protocols is the PCR amplification step, the error rate of which should be sufficiently low not to overshadow the mutations that should be detected. In a comparative
study it was found that the PCR error rate could be greatly reduced by using high-fidelity polymerases, such as T4 and T7, and by alterations of the experimental conditions (Keohavong and Thilly, 1989). Cariello et al. (1991) examined the fidelity of several DNA polymerases used in PCR and found Vent polymerase to cause the least amount of errors, i.e. 2.4 × l0 -5. This is still too high, however, to make direct mutation analysis without selection a feasible strategy. An interesting method was developed by Sandy et al. 0992). In this system, point mutations in Taq I restriction sites are detected by the selective amplification of mutated recognition sequences; the mutant fragments rescued in this way are directly sequenced or identified by oligonucleotide hybridization. Because of the extremely small mutational target the risk of PCR-related errors is small, although some were observed (Sandy et al., 1992). However, this very small mutational target is also the method's greatest disadvantage; although it allows the measurement of spontaneous mutations in human
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Somatic mutations and cellular aging and animal tissues without selection, it may not be very representative for the total of mutational events occurring with age. SUMMARY AND GENERAL DISCUSSION
The major conclusion that can be drawn from this overview on somatic mutations and cellular aging is that the increased mutation load predicted from the inherent imperfection of DNA maintenance and repair systems seems to be present. For chromosomal aberrations the majority of reports indicate increased levels with age. The same is true for mutations present in HPRT and HLA-A in human blood cells. This, however, does not give us a clear picture of the mutation load in different organs and tissues. Neither does it provide information about the kinetics of mutation accumulation. The accumulation of mutations in a linear fashion is not likely to lead to extremely high levels at old age and would therefore suggest mutation accumulation to be a consequence rather than a cause of aging. However, an exponential increase, for example, due to a decrease in the proficiency of cellular defense (e.g. DNA repair, anti-oxidant systems), could lead to extremely high levels of mutations at old age. The very slow emergence of hard data on mutation accumulation with age has been accompanied, especially during the last couple of years, by the development of methods that allow mutation detection and characterization at a large scale. In the context of ongoing studies toward that aim, it could be useful to speculate about how mutations contribute to aging. In this regard one can ask the following question: at which frequency and at which loci should mutations occur to cause the specific age-related degenerations that can be observed? The answer clearly should take into account the possibility that mutant somatic cells can be lost from the population, either as a consequence of the mutation itself or by programmed cell death as a sort of self-protection against too high a mutation load. Selection is a good explanation for the apparent immortality of the germ-line; even after conception approximately 15-20% of pregnancies terminate in spontaneous abortion as a consequence of chromosomal anomalies (Plachot et al., 1987; Minguillon et al., 1989; see, also, Vijg, 1990). Although such selection leading to cell death will certainly occur after the occurrence of mutational events in crucial (housekeeping) genes, the question remains as to what would happen when mutational events only result in slightly changed gene expression levels. In cell cultures this would most likely lead to growth (dis)advantages of particular cells and hence the retention of uniformity. In multicellular organisms, however, the static distribution of cells over a great variety of organs and tissues would result in a mosaic, much like the situation in certain single plants (Sutherland and Watkinson, 1986). The question then
remains as to how much genetic variability would be necessary to explain age-related functional decline and death. Two lines of research should, within several years, provide the answers. First, accurate data on mutation loads in different organs and tissues of the recently generated transgenic mouse models can be expected soon. Such data can then be correlated with patterns of age-related multiple pathology and particular forms of age-related functional decline. Second, mathematical modeling using the wealth of information on the regulatory control of many genes in the human and animal genomes that are presently generated, may provide some answers to the question of how much mutations and in what sequences are necessary to cause particular agerelated defects. REFERENCES
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