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Mutation theory of aging, assessed in transgenic mice and knockout mice
Mechanisms of Ageing and Development 123 (2002) 1543 /1552 www.elsevier.com/locate/mechagedev
Mutation theory of aging, assessed in transgenic mice and knockout mice Tetsuya Ono , Yoshihiko Uehara, Yusuke Saito, Hironobu Ikehata Department of Cell Biology, Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Abstract A vital question in the mutation theory of aging is whether mutation accumulates with age. If it does, what are the causes and consequences of the accumulation of mutation? The recent development of transgenic mice has made it possible to study mutation in different kinds of tissues and at a molecular level. An application of these mice to the study of age-dependent alteration has revealed that mutation does accumulate in the aging process. Studies have also revealed several important characteristics of mutation associated with aging. (1) The rate of age-dependent increase of mutant frequency varies among different types of tissue. (2) The rate is not in parallel with the cell proliferation rate of the tissue. (3) Some types of mutation are unique to specific tissues, suggesting the presence of a mechanism of mutation relative to tissue type. On the other hand, several kinds of knockout mice defective in DNA repair have been shown to exhibit tissue lesions and shortened life span. These characteristics provide a new view on the relationship between aging and the genome maintenance system. Here we review the current status of research on the correlation between mutation and aging undertaken by the use of transgenic and knockout mice. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mutation; Aging; Transgenic mouse; Knockout mouse
1. Introduction Several decades ago radiation was demonstrated to shorten the life span of mice in a dosedependent manner (Lundop and Rotblat, 1961). Since a major biological effect of radiation was likely to be DNA damage, it was natural to imagine that radiation-induced alteration in DNA plays a causal role in life span shortening. Corresponding author. Tel.: /81-22-717-8131; fax: /8122-717-8136 E-mail address: [email protected] (T. Ono).
Subsequently, living systems were proven to have a complex system to repair DNA damage. The system, however, is not infallible, and can make mistakes that result in alteration */i.e. mutation */ of DNA sequences. In fact, most agents that alter the chemical structure of DNA in cells elicit mutation. The present picture we have of the DNA molecule in living cells is thus dynamic (Fig. 1). The DNA suffers continuous damage from exogenous agents such as background radiation, UV light, and chemicals, and also from endogenous agents such as reactive oxygen and radicals created through metabolism in cells.
0047-6374/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 2 ) 0 0 0 9 0 - 8
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Fig. 1. Schematic illustration of mutation formation. Doublestranded DNA in living cells is constantly exposed to many kinds of damaging agents, but most of the lesions are repaired. DNA damage as well as mistakes in DNA repair and DNA replication lead to formation of mutation.
Concomitantly, the damage is repaired by different kinds of repair systems (Hoeijmakers, 2001). The estimated number of repair genes in the human genome is 130 (Wood et al., 2001). These dynamics will result in an accumulation of mistakes over a long period of time such as that involved in the aging process. DNA replication is also shown to be imperfect, and mistakes arise. Thus, it is easy to imagine that mutation accumulates in the aging process. The important characteristics of mutations are that they are irreversible and in many cases hazardous to cells. That the accumulation of mutations will become toxic to cells and multi-cellular systems is the basic concept behind a mutation theory of aging (Kirkwood, 1989; Finch, 1990; Vijg, 2000). Many lines of evidence support this theory (Finch, 1990; Vijg, 2000). DNA damage has been shown to be increased in aged animals and humans. The frequency of chromosomal abnormality is shown to be elevated with age. However, these data contrast with negative results showing no ageassociated change (Finch, 1990). The examination of long-lived mutants of Caenorhabditis elegans revealed that it has an elevated defense system against the external stress from sources such as UV light, oxygen, and heat (Murakami and Johnson, 1996; Johnson et al., 2000). Those findings suggest the importance of a self-defense mechanism in establishment of a long life. Suppression of mutation induction through efficient DNA repair is one of the main self-defense systems. More recently, the mutation theory has been challenged directly
by examination of mutation itself. Since mutation occurs at very low frequency (e.g. 104 or 106), the usual biochemical methods are not applicable. This problem has been overcome by the development of transgenic mice feasible for mutation analysis (Vijg et al., 1997; Nohmi et al., 2000). The mice contain bacterial genes so that the mutation of the genes can be examined by a transfer of the genes to bacterial systems. Up to now, three strains of transgenic mice has been utilized for aging studies. The results are reviewed here. Another kind of challenge to the mutation theory has been launched. It involves destruction of a specific gene in the mouse. The mouse is called a knock-out (KO) mouse. Some KO mice having a deficiency in one of the DNA repair genes show a high mutation frequency and tissue lesions including cancer. These findings support the mutation theory of aging. Scrutiny of the data, however, indicates that the relationship is not so simple. The papers on these mice are reviewed in the latter half of this article.
2. Do mutations accumulate in the aging process Age-dependent increase of mutation frequency was observed previously in blood lymphocytes and erythrocytes of human and mouse (Branda et al., 1993; Robinson et al., 1994; Hirai et al., 1995; Kyoizumi et al., 1996). Highly elevated mutation frequency was also observed in lymphocytes of senescence-accelerated mice (SAM) (Odagiri et al., 1998). These results, however, are difficult to apply to other kinds of cells in vivo because the lymphocytes and erythrocytes have unique characteristics in vivo. They are terminally differentiated and destined to disappear after certain period of time through cellular turnover. Genome maintenance systems in these cell lineages, especially at later stages of differentiation, might be different from those working in cells which keep on functioning for a long period of time with no or scarce cellular turnover. Thus, a direct measurement of mutation in different kinds of tissues would have critical meaning.
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The study of mutation in various tissues became possible only after the development of transgenic mice suitable for mutation assay. The age-dependencies of mutation frequency examined thus far are summarized in Table 1. Three strains of mice were used: MutaTM mouse harboring lacZ -containing l DNA, Line 60 mouse harboring lacZ containing plasmid DNA, and Big-BlueTM mouse harboring lacI -containing l DNA. In Table 1, the rates of increase were classified into four groups: /, no increase; /, less than 2-fold increase during 2 years of life; //, 2 /3-fold increase during 2 years; ///, more than 3-fold increase during 2 years. We did not include the data on changes up to 1 year of age. From these results, the following three characteristics could be deduced. 1) Mutation frequency increased with age in many types of tissue examined. 2) The rate of increase varied among the types of tissue. The highest rate of increase was observed in the small intestine and urinary bladder. 3) The rate of increase did not show parallelism with the cellular proliferation characteristic of the tissue. For example, mutation accumulations in skin and testis, both of which contain highly proliferating cells, revealed low rates of increase, while those in the heart and liver, organs with non- or slowly-proliferating tissue, were high. These findings seem to suggest
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that a major cause of mutation accumulation is not related to errors in DNA replication. Observation showed some discrepancies among the mouse strains, mainly in testicular cells and the urinary bladder. These findings might reflect a difference in the chromosomal location where the three transgenes are integrated among these strains of mouse. Another possible explanation for these differences could be differences in food and environment, given that the animals were maintained in different labs. At present, it is difficult to specify the cause. It is interesting to note that the mutation frequency in newborn or young mice was similar among the different types of tissue. Hence, the tissue-specific variation in old mice must be established in the aging process after maturation.
3. What is the cause of mutation accumulation in the aging process Some mutagens are now known to induce unique types of mutation. For example, reactive oxygen induces a base substitution of G:C to T:A transversion type mutation through the formation of 8-oxoguanine and its mispairing to A (instead of C) (Hoeijmakers, 2001). Thus, elucidation of the molecular nature of mutations accumulated in old mice might yield clues on the cause of
Table 1 Age-dependent increase of spontaneous mutant frequency in three strains of transgenic mice Tissue
Mutaa
Spleen Liver Brain Heart Skin Small Intestine Urinary Bladder Testis
// (Ono et al., 2000) // (Ono et al., 2000) / (Ono et al., 2000) // (Ono et al., 2000) / (Ono et al., 2000) /// (Ono, unpublished data) / (Ono, unpublished data) / (Ono et al., 2000)
Line60b
/ (Dolle´ et al., 1997) / (Dolle´ et al., 1997) // (Dolle´ et al., 2000)
Big-Bluec // (Lee et al., 1994) // (Stuart et al., 2000) / (Stuart et al., 2000)
/// (Dolle´ et al., 2000) / (Martin et al., 2001)
/// (Stuart et al., 2000) /// (Walter et al., 1998)
Mutation increase was calculated by dividing mutant frequency at 2 years of age by that at 2 months of age, and classified as follows: /, no increase; /, an increase of less than 2-fold; //, an increase of 2 /3-fold; ///, an increase of more than 3-fold. (), reference. a Muta mouse contains lacZ gene wrapped up in lambda genome. b Line60 mouse contains lacZ gene in a plasmid construct. c Big-Blue mouse contains lacI gene wrapped up in lambda genome.
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mutation. Studies of the molecular nature of mutations have been carried out by several groups using the three mouse stains listed in Table 1. The results obtained thus far are summarized as follows. 1) Distribution of the frequencies of different types of mutation, referred to as the mutation spectrum, is similar among tissue types and does not change much in the aging process. For example, data on the skin and heart of MutaTM mouse is shown in Fig. 2. The most frequent change is a base substitution of the G:C to A:T transition at the DNA sequence of 5?CG3?. In Line 60 mice, a large deletion is the most abundant alteration. This type of mutation cannot be detected in MutaTM and Big-
BlueTM mice due to a technical limitation (Vijg et al., 1997; Nohmi et al., 2000), though a large deletion of DNA could be the most common type of mutation. Further studies are needed to elucidate this point. It was concluded that in the three mouse strains, the main cause(s) of mutation seems to remain similar in the aging process in most types of tissue. One exception is the small intestine in old mice (Dolle´ et al., 2000). 2) Some types of mutation have been elucidated to accumulate with age in specific tissues. In liver, two simultaneous base substitutions in neighboring positions, called a tandem mutation, accumulate with age and constitute 10 / 15% of the total number of mutations in old mouse (Buettner et al., 1999; Ono et al., 2000). As a cause of tandem mutation, two types of mutagens have been reported; ultraviolet light and DNA cross-linking agents such as aldehyde (Hutchinson, 1994; Matsuda et al., 1998). Since ultraviolet light cannot reach the liver because of very poor penetration through the body, cross-linking chemicals could be the agent responsible for the tandem mutation observed in old liver. Buettner et al. observed the age-dependent accumulation of a tandem mutation in adipose tissue (Buettner et al., 1999). In old small intestine, all kinds of base substitutions accumulate and predominate other types of mutation (Dolle´ et al., 2000). The small intestine might have weak repair activity for base mismatches, or it might be exposed to different kinds of highly mutagenic substances. These lines of evidence indicate the presence of tissue-specific causes for age-dependent accumulation of mutation. It is not clear whether this reflects differences in DNA damaging agents or in DNA repair systems.
Fig. 2. Spectrum of different types of spontaneous mutation observed in skin (A) and heart (B) of newborn and old lacZ transgenic mice. The frequencies of each type of mutation in newborn (grey column) and 23 months old (black column) mice are compared side by side. The figure is based on the data of Ono et al. (2000).
4. What are the consequences of a high level of mutation Mutation theory assumes that a high level of mutation deteriorates cell as well as tissue func-
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tions. However, it has not yet been determined if this is really the case. Even if it is, the question of what level of mutation is deleterious remains to be answered. A recent development in creating KO mice could provide a clue in this regard. The several KO mouse strains deficient in one of the DNA repair genes have been shown to sustain elevated levels of mutation and/or some defects in tissue function. These strains and mutations/defects are listed in Table 2. The repair systems that have been inactivated by destruction of a gene include mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ), recombination and telomere maintenance. We did not include KO mice of topoisomerase IIIb (Kwan and Wang, 2001) in the Table because the role of this system in vivo is not yet clear and it could be involved in something other than DNA repair. It is noted that the topoisomerase IIIb KO mice have a short life span. Although most of the mutation studies done using the KO mice listed in Table 2 are involved young mice, the mutation frequencies were elevated in several mice. The absence of elevation of mutation in some mice can be explained easily by the possible existence of other repair pathway(s) operating on the same DNA damage. The inactivation of the three genes involved in MMR, Mlh1 , Pms2 , and Msh2 , raised mutation in many types of tissue to extremely high levels and resulted in the early occurrence of tumor in the thymus or intestine. The mutation frequencies in the brain and heart of Msh2 (///) mice have been shown to be elevated. However, the early death of the mice makes it difficult to assess whether the elevation of mutation in brain and heart accelerates aging processes in the tissues. Mutation frequency in the liver of Ogg1 -deficient mice showed about a 2-fold elevation at a young age (Klungland et al., 1999; Minowa et al., 2000). The livers of these mice also contain a high level of 8-oxoguanine in the genomic DNA. The histological studies, however, showed no lesions up to at least 1 year of age. These findings may suggest that a slight elevation of mutation can be
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tolerable for tissues in vivo. Another possibility is that some lesions could become overt later in the aging process, perhaps at older ages than 1 year. Further study is necessary to elucidate this point. Findings in a recent study on Xp c(///) mice by Wijnhoven et al. (2000), who found that the Xpc KO mice did not show any elevation of mutation at a young age but showed 30 times as many mutations in splenic T cells at 12 and 18 months of age, are of interest. In spite of the high mutation rate, the mice did not reveal any elevation of tumor incidence or accelerated aging in the tissues. Those results might indicate that a high rate of mutation is tolerable in some cases and does not result in any tissue lesion. However, it is not clear whether the elevation of mutation is limited to T-cells or is common to other tissues. Further studies on different types of tissue as well as on different kinds of NER-deficient mice will provide important clues. On the other hand, Ercc1- and Ku86 -deficient mice showed early onset of age-associated alterations in many types of tissue (Weeda et al., 1997; Vogel et al., 1999). Although mutations in these mice have not yet been studied, caution is essential in interpreting the results obtained using these mice. The alteration observed might not be directly related to the deficiency in DNA repair. For example, Ku86 is known to be involved in establishment of immunological diversity of lymphocytes. Thus, the tissue damage in adult mice could be a consequence of immuno-deficiency rather than of DNA repair-deficiency. The Ercc1-deficient mice also show a high rate of embryonic lethality, reduced body weight at birth, and lethality at about 1 month of age. The senescent phenotypes observed in many types of tissue from the mice could be a result of developmental failure induced by the Ercc1 -deficiency rather than by mutation. The reason for why development is affected by the gene destruction is unclear. The results in Blm KO mice and telomerasedeficient mice indicate that the destruction of chromosomal structure could also lead to accelerated aging (Rudolph et al., 1999; Luo et al., 2000).
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Table 2 Effects of repair gene inactivation on mutation and tissue lesion Inactivated gene
Type of repair involveda
Mutation (Marker)b (Tissues)
Mlh1
Pms2
MMR
MMR
Old r
/// (microsatellite, Ouab ) (small intestine, fibroblasts) /// (SupF, lacI) (small intestine, colon, skin) /// (micrtosatellite, Ouabr) Lymphoma (small intestine, fibroblasts) /// (SupF, lacI) (small intestine, colon, skin) /// (microsatellite, lacI) (small intestine, thymus, brain, heart)
Tissue lesion Tumor in intestine
Yao et al., 1999; Baross-Francis et al., 2001 Prolla et al., 1998
Msh2
MMR
Msh6 Ogg1
MMR BER
/ (microsatellite) (tumor) / (gpt, lacI) (liver)
Tumor in intestine, lymphoma (increase of 8-oxyguanine in DNA)
Ung Xpa
BER NER
/ (lacI) (spleen, thymus) / (rpsL) (skin)
(increase of U in DNA) Increased UV sensitivity
Xpc Ercc1
NER NER
Ku86
NHEJ
Blm mTR
Recombination Telomere maintenance
a b
/ (HPRT) (splenic T-cell)
/// (LOH at HPRT)
/ (lacZ) (liver) / (HPRT) (splenic T-cell) /// (HPRT)
Prolla et al., 1998
Thymoma
Increased UV sensitivity Delay in development and growth, lesions in many tissues Delay in growth, lesions in bone, skin and liver High incidence of various kinds of tumors Acceleration of many aging phenotypes in three to six generations in parallel with telomere shortening
MMR, mismatch repair; BER, base excision repair; NER, nucleotide excision repair; NHEJ, non-homologous end joining. /, same level as wild type; /, an increase of less than 3-fold; ///, an increase of more than 10-fold.
Yao et al., 1999; Baross-Francis et al., 2001 Wind et al., 1995; Andrew et al., 1998; Baross-Francis et al., 1998 Edelmann et al., 1997 Klungland et al., 1999; Minowa et al., 2000 Nilsen et al., 2000 Murai et al., 2000 Giese et al., 1999 Wijnhoven et al., 2000 Wijnhoven et al., 2000 Weeda et al., 1997 Vogel et al., 1999 Luo et al., 2000 Rudolph et al., 1999
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Young
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Determination of the exact molecular mechanisms is pending. Overall, the study of the correlations between mutation and phenotypic alteration in tissues in DNA repair gene KO mice is still in the preliminary stages. Further study will provide data that will prove or disprove the mutation theory of aging.
5. Future studies The studies reviewed above clearly indicate that mutation in the aged mouse is tissue-specific. Since mutation frequency as well as mutation spectrum seem to be similar among the tissues in newborn and young mice, the mechanism behind the tissuespecificity must become operational in the aging process after maturation. A major cause of ageassociated accumulation of mutation does not seem to be related to an error associated with DNA replication, based on the fact that non- or slowly-proliferating tissues such as that in the heart and liver show a rate of age-associated accumulation of mutation higher than that of highly proliferating tissues such as the skin and testis. What, then, might be the cause of these mutation accumulation? The cause could be tissuespecific DNA damage or DNA repair capability. At present, it is difficult to say which is more important. Possible approaches would be to elevate or reduce mutation by artificial manipulation. Recently, radiation has been proved to induce mutation in many tissues in the mouse (Ono et al., 1999; Nakamura et al., 2000). The molecular nature of mutation induced by radiation, however, appears to be different from those observed in the tissues of old mouse. This finding indicates that natural background radiation is not the major cause of mutation that accumulates in the aging process. The rate of radiation-induced mutation in the brain is about one-half that in the spleen (Nakamura et al., 2000), and corresponds to the low rate of age-related mutation accumulation in the brain when compared with that in the spleen (Table 1). It
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has previously been demonstrated that the number of DNA breaks induced by radiation as well as their repair rates are similar between proliferating and non-proliferating tissues (Ono and Okada, 1974, 1978). These data might suggest that the repair system in the brain is more accurate for some reason. Similar analyses using different kinds of chemical mutagens and reactive oxygens will provide clues for understanding the cause of the mutation accumulating in the aging process. At the same time, the possible importance of the DNA repair system should be established. We do not know whether the DNA repair system alters with age or whether it differs among various types of tissue. Expression of each DNA repair gene should be examined in individual tissues throughout the aging process. Each type of tissue might have a unique DNA protection system, as has been suggested by radiation-induced mutation studies (Nakamura et al., 2000). Among the many kinds of DNA repair genes, the thymine DNA glycosylase gene (Tdg ) would be interesting to investigate because it has been proved to be involved in a repair process responsible for the G:C to A:T mutation at the 5?CG3? sequence, which is the most frequent mutation observed in many types of tissue (Wiebauer and Jiricny, 1989). This type of mutation is assumed to be created by three steps (Gonzalgo and Jones, 1997): (i) methylation of cytosine at position 5 in the 5?CG3? sequence
Fig. 3. Schematic illustration of a formation of G:T mismatch at the 5?CG3? DNA sequence, which leads to base substitution of C:G to T:A. mC indicates 5 methylcytosine. TDG is thymine DNA glycosylase. The mutagenic T:G mismatch is repaired by TDG.
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resulting in 5-methylcytosine formation, (ii) spontaneous deamination at position 4 of 5-methylcytosine, which results in formation of T and T:G mispair instead of C:G, and (iii) replication of DNA leading to T:A and C:G pairing in the two daughter strands (Fig. 3). In mammals, the T:G mispairing could be repaired by TDG. If this is the case in tissues, an elevation of this enzyme level should reduce mutation frequency. Conversely, an inactivation of the Td g gene should raise mutation frequency. Our preliminary study on the Tdg gene KO mice, however, showed lethality of the mouse at the embryonic stage (Saito et al., 2001). Similar phenomena have been observed with destruction of the other repair genes (Friedberg and Meira, 1999). These findings indicate that some of the DNA repair proteins have a vital importance at the embryonic stage. In these cases, the significance of the protein in the aging process could be elucidated only if the responsible gene is inactivated after maturation. This inactivation can be achieved by applying the technique of conditional gene knockout (Lewandoski, 2001). Although this technique is as yet incomplete, inactivation of a specific gene in a certain tissue at a desired time will become feasible in the near future. The application of this method will assist us in separating the embryonic stage from the aging process and also in modifying genes in each type of tissue. To this end, we should be attentive to the development of new biotechnologies.
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Ono, T., Okada, S., 1978. Does the capacity to rejoin radiationinduced DNA breaks decline in senescent mice. Int. J. Radiat. Biol. 33, 403 /407. Ono, T., Ikehata, H., Nakamura, S., Saito, Y., Komura, J., Hosoi, Y., Yamamoto, K., 1999. Molecular nature of mutations induced by a high dose of X-rays in spleen, liver, and brain of the lacZ -transgenic mouse. Environ. Mol. Mutagen. 34, 97 /105. Ono, T., Ikehata, H., Nakamura, S., Saito, Y., Hosoi, Y., Takai, Y., Yamada, S., Onodera, J., Yamamoto, K., 2000. Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ transgenic mouse. Mutation Res. 447, 165 /177. Prolla, T.A., Baker, S.M., Harris, A.C., Tsao, J.L., Yao, X., Bronner, C.E., Zheng, B., Gordon, M., Reneker, J., Arnheim, N., Shibata, D., Bradley, A., Liskay, R.M., 1998. Tumour susceptibility and spontaneous mutation in mice deficient in Hlh1, Pms1 and Pms2 DNA mismatch repair. Nat. Gen. 18, 276 /279. Robinson, D.R., Goodall, K., Albertini, R.J., O’Neill, J.P., Finette, B., Sala-Trepat, M., Moustacchi, E., Tates, A.D., Beare, D.M., Green, M.H.L., Cole, J., 1994. An analysis of in vivo hprt mutant frequency in circulating T-lymphocytes in the normal human population: a comparison of four datasets. Mutation Res. 313, 227 /247. Rudolph, K.L., Chang, S., Lee, H.-W., Blasco, M., Gottlieb, G.J., Greider, C., DePinho, R.A., 1999. Longevity, stress response, and cancer in aging telomerase-deficient mice. Cell 96, 701 /712. Saito, Y., Uehara, Y., Takeda, N., Suzuki, M., Nohmi, T., Noda, T., Ono, T., 2001. The effect of thymine DNA glycosylase deficiency in mice. Mutation Res. 483 (Suppl. 1), S126. Stuart, G.R., Oda, Y., de Boer, J.G., Glickman, B.W., 2000. Mutation frequency and specificity with age in liver, bladder and brain of lacI transgenic mice. Genetics 154, 1291 /1300. Vijg, J., 2000. Somatic mutations and aging: a re-evaluation. Mutation Res. 447, 117 /135. Vijg, J., Dolle, M.E.T., Martus, H.J., Boerrigter, M.E.T.I., 1997. Transgenic mouse models for studying mutations in vivo: applications in aging research. Mech. Age. Develop. 98, 189 /201. Vogel, H., Lim, D.S., Karsenty, G., Finegold, M., Hasty, P., 1999. Deletion of Ku86 causes early onset of senescence in mice. Proc. Natl. Acad. Sci. USA 96, 10770 /10775. Walter, C.A., Intano, G.W., McCarrey, J.R., McMahan, C.A., Walter, R.B., 1998. Mutation frequency declines during spermatogenesis in young mice but increases in old mice. Proc. Natl. Acad. Sci. USA 95, 10015 /10019. Weeda, G., Donker, I., de Wit, J., Morreau, H., Janssens, R., Vissers, C.J., Nigg, A., van Steeg, H., Bootsma, D., Hoeijmakers, J.H.J., 1997. Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence. Curr. Biol. 7, 427 /439. Wiebauer, K., Jiricny, J., 1989. In vitro correction of GT mispairs to GC pairs in nuclear extracts from human cells. Nature 339, 234 /236.
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Wijnhoven, S.W.P., Kool, H.J.M., Mullenders, L.H.F., van Zeeland, A.A., Friedberg, E.C., van der Horst, G.T.J., van Steeg, H., Vrieling, H., 2000. Age-dependent spontaneous mutagenesis in Xpc mice defective in nucleotide excision repair. Oncogene 19, 5034 /5037. Wind, N., Dekker, M., Berns, A., Radman, M., Riele, H., 1995. Inactivation of the mouse Msh2 gene results in mismatch repair deficiency, methylation tolerance, hyperrecombination, and predisposition to cancer. Cell 82, 321 /330.
Wood, R.D., Mitchell, M., Sgouros, J., Lindahl, T., 2001. Human DNA repair genes. Science 291, 1284 / 1289. Yao, X., Buermeyer, A.B., Narayanan, L., Tran, D., Baker, S.M., Prolla, T.A., Glazer, P.M., Liskay, R.M., Arnheim, N., 1999. Different mutator phenotypes in Mlh1-versus Pms2-deficient mice. Proc. Natl. Acad. Sci. USA 96, 6850 / 6855.
Mutation theory of aging, assessed in transgenic mice and knockout mice Tetsuya Ono , Yoshihiko Uehara, Yusuke Saito, Hironobu Ikehata Department of Cell Biology, Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan
Abstract A vital question in the mutation theory of aging is whether mutation accumulates with age. If it does, what are the causes and consequences of the accumulation of mutation? The recent development of transgenic mice has made it possible to study mutation in different kinds of tissues and at a molecular level. An application of these mice to the study of age-dependent alteration has revealed that mutation does accumulate in the aging process. Studies have also revealed several important characteristics of mutation associated with aging. (1) The rate of age-dependent increase of mutant frequency varies among different types of tissue. (2) The rate is not in parallel with the cell proliferation rate of the tissue. (3) Some types of mutation are unique to specific tissues, suggesting the presence of a mechanism of mutation relative to tissue type. On the other hand, several kinds of knockout mice defective in DNA repair have been shown to exhibit tissue lesions and shortened life span. These characteristics provide a new view on the relationship between aging and the genome maintenance system. Here we review the current status of research on the correlation between mutation and aging undertaken by the use of transgenic and knockout mice. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Mutation; Aging; Transgenic mouse; Knockout mouse
1. Introduction Several decades ago radiation was demonstrated to shorten the life span of mice in a dosedependent manner (Lundop and Rotblat, 1961). Since a major biological effect of radiation was likely to be DNA damage, it was natural to imagine that radiation-induced alteration in DNA plays a causal role in life span shortening. Corresponding author. Tel.: /81-22-717-8131; fax: /8122-717-8136 E-mail address: [email protected] (T. Ono).
Subsequently, living systems were proven to have a complex system to repair DNA damage. The system, however, is not infallible, and can make mistakes that result in alteration */i.e. mutation */ of DNA sequences. In fact, most agents that alter the chemical structure of DNA in cells elicit mutation. The present picture we have of the DNA molecule in living cells is thus dynamic (Fig. 1). The DNA suffers continuous damage from exogenous agents such as background radiation, UV light, and chemicals, and also from endogenous agents such as reactive oxygen and radicals created through metabolism in cells.
0047-6374/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 4 7 - 6 3 7 4 ( 0 2 ) 0 0 0 9 0 - 8
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Fig. 1. Schematic illustration of mutation formation. Doublestranded DNA in living cells is constantly exposed to many kinds of damaging agents, but most of the lesions are repaired. DNA damage as well as mistakes in DNA repair and DNA replication lead to formation of mutation.
Concomitantly, the damage is repaired by different kinds of repair systems (Hoeijmakers, 2001). The estimated number of repair genes in the human genome is 130 (Wood et al., 2001). These dynamics will result in an accumulation of mistakes over a long period of time such as that involved in the aging process. DNA replication is also shown to be imperfect, and mistakes arise. Thus, it is easy to imagine that mutation accumulates in the aging process. The important characteristics of mutations are that they are irreversible and in many cases hazardous to cells. That the accumulation of mutations will become toxic to cells and multi-cellular systems is the basic concept behind a mutation theory of aging (Kirkwood, 1989; Finch, 1990; Vijg, 2000). Many lines of evidence support this theory (Finch, 1990; Vijg, 2000). DNA damage has been shown to be increased in aged animals and humans. The frequency of chromosomal abnormality is shown to be elevated with age. However, these data contrast with negative results showing no ageassociated change (Finch, 1990). The examination of long-lived mutants of Caenorhabditis elegans revealed that it has an elevated defense system against the external stress from sources such as UV light, oxygen, and heat (Murakami and Johnson, 1996; Johnson et al., 2000). Those findings suggest the importance of a self-defense mechanism in establishment of a long life. Suppression of mutation induction through efficient DNA repair is one of the main self-defense systems. More recently, the mutation theory has been challenged directly
by examination of mutation itself. Since mutation occurs at very low frequency (e.g. 104 or 106), the usual biochemical methods are not applicable. This problem has been overcome by the development of transgenic mice feasible for mutation analysis (Vijg et al., 1997; Nohmi et al., 2000). The mice contain bacterial genes so that the mutation of the genes can be examined by a transfer of the genes to bacterial systems. Up to now, three strains of transgenic mice has been utilized for aging studies. The results are reviewed here. Another kind of challenge to the mutation theory has been launched. It involves destruction of a specific gene in the mouse. The mouse is called a knock-out (KO) mouse. Some KO mice having a deficiency in one of the DNA repair genes show a high mutation frequency and tissue lesions including cancer. These findings support the mutation theory of aging. Scrutiny of the data, however, indicates that the relationship is not so simple. The papers on these mice are reviewed in the latter half of this article.
2. Do mutations accumulate in the aging process Age-dependent increase of mutation frequency was observed previously in blood lymphocytes and erythrocytes of human and mouse (Branda et al., 1993; Robinson et al., 1994; Hirai et al., 1995; Kyoizumi et al., 1996). Highly elevated mutation frequency was also observed in lymphocytes of senescence-accelerated mice (SAM) (Odagiri et al., 1998). These results, however, are difficult to apply to other kinds of cells in vivo because the lymphocytes and erythrocytes have unique characteristics in vivo. They are terminally differentiated and destined to disappear after certain period of time through cellular turnover. Genome maintenance systems in these cell lineages, especially at later stages of differentiation, might be different from those working in cells which keep on functioning for a long period of time with no or scarce cellular turnover. Thus, a direct measurement of mutation in different kinds of tissues would have critical meaning.
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The study of mutation in various tissues became possible only after the development of transgenic mice suitable for mutation assay. The age-dependencies of mutation frequency examined thus far are summarized in Table 1. Three strains of mice were used: MutaTM mouse harboring lacZ -containing l DNA, Line 60 mouse harboring lacZ containing plasmid DNA, and Big-BlueTM mouse harboring lacI -containing l DNA. In Table 1, the rates of increase were classified into four groups: /, no increase; /, less than 2-fold increase during 2 years of life; //, 2 /3-fold increase during 2 years; ///, more than 3-fold increase during 2 years. We did not include the data on changes up to 1 year of age. From these results, the following three characteristics could be deduced. 1) Mutation frequency increased with age in many types of tissue examined. 2) The rate of increase varied among the types of tissue. The highest rate of increase was observed in the small intestine and urinary bladder. 3) The rate of increase did not show parallelism with the cellular proliferation characteristic of the tissue. For example, mutation accumulations in skin and testis, both of which contain highly proliferating cells, revealed low rates of increase, while those in the heart and liver, organs with non- or slowly-proliferating tissue, were high. These findings seem to suggest
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that a major cause of mutation accumulation is not related to errors in DNA replication. Observation showed some discrepancies among the mouse strains, mainly in testicular cells and the urinary bladder. These findings might reflect a difference in the chromosomal location where the three transgenes are integrated among these strains of mouse. Another possible explanation for these differences could be differences in food and environment, given that the animals were maintained in different labs. At present, it is difficult to specify the cause. It is interesting to note that the mutation frequency in newborn or young mice was similar among the different types of tissue. Hence, the tissue-specific variation in old mice must be established in the aging process after maturation.
3. What is the cause of mutation accumulation in the aging process Some mutagens are now known to induce unique types of mutation. For example, reactive oxygen induces a base substitution of G:C to T:A transversion type mutation through the formation of 8-oxoguanine and its mispairing to A (instead of C) (Hoeijmakers, 2001). Thus, elucidation of the molecular nature of mutations accumulated in old mice might yield clues on the cause of
Table 1 Age-dependent increase of spontaneous mutant frequency in three strains of transgenic mice Tissue
Mutaa
Spleen Liver Brain Heart Skin Small Intestine Urinary Bladder Testis
// (Ono et al., 2000) // (Ono et al., 2000) / (Ono et al., 2000) // (Ono et al., 2000) / (Ono et al., 2000) /// (Ono, unpublished data) / (Ono, unpublished data) / (Ono et al., 2000)
Line60b
/ (Dolle´ et al., 1997) / (Dolle´ et al., 1997) // (Dolle´ et al., 2000)
Big-Bluec // (Lee et al., 1994) // (Stuart et al., 2000) / (Stuart et al., 2000)
/// (Dolle´ et al., 2000) / (Martin et al., 2001)
/// (Stuart et al., 2000) /// (Walter et al., 1998)
Mutation increase was calculated by dividing mutant frequency at 2 years of age by that at 2 months of age, and classified as follows: /, no increase; /, an increase of less than 2-fold; //, an increase of 2 /3-fold; ///, an increase of more than 3-fold. (), reference. a Muta mouse contains lacZ gene wrapped up in lambda genome. b Line60 mouse contains lacZ gene in a plasmid construct. c Big-Blue mouse contains lacI gene wrapped up in lambda genome.
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mutation. Studies of the molecular nature of mutations have been carried out by several groups using the three mouse stains listed in Table 1. The results obtained thus far are summarized as follows. 1) Distribution of the frequencies of different types of mutation, referred to as the mutation spectrum, is similar among tissue types and does not change much in the aging process. For example, data on the skin and heart of MutaTM mouse is shown in Fig. 2. The most frequent change is a base substitution of the G:C to A:T transition at the DNA sequence of 5?CG3?. In Line 60 mice, a large deletion is the most abundant alteration. This type of mutation cannot be detected in MutaTM and Big-
BlueTM mice due to a technical limitation (Vijg et al., 1997; Nohmi et al., 2000), though a large deletion of DNA could be the most common type of mutation. Further studies are needed to elucidate this point. It was concluded that in the three mouse strains, the main cause(s) of mutation seems to remain similar in the aging process in most types of tissue. One exception is the small intestine in old mice (Dolle´ et al., 2000). 2) Some types of mutation have been elucidated to accumulate with age in specific tissues. In liver, two simultaneous base substitutions in neighboring positions, called a tandem mutation, accumulate with age and constitute 10 / 15% of the total number of mutations in old mouse (Buettner et al., 1999; Ono et al., 2000). As a cause of tandem mutation, two types of mutagens have been reported; ultraviolet light and DNA cross-linking agents such as aldehyde (Hutchinson, 1994; Matsuda et al., 1998). Since ultraviolet light cannot reach the liver because of very poor penetration through the body, cross-linking chemicals could be the agent responsible for the tandem mutation observed in old liver. Buettner et al. observed the age-dependent accumulation of a tandem mutation in adipose tissue (Buettner et al., 1999). In old small intestine, all kinds of base substitutions accumulate and predominate other types of mutation (Dolle´ et al., 2000). The small intestine might have weak repair activity for base mismatches, or it might be exposed to different kinds of highly mutagenic substances. These lines of evidence indicate the presence of tissue-specific causes for age-dependent accumulation of mutation. It is not clear whether this reflects differences in DNA damaging agents or in DNA repair systems.
Fig. 2. Spectrum of different types of spontaneous mutation observed in skin (A) and heart (B) of newborn and old lacZ transgenic mice. The frequencies of each type of mutation in newborn (grey column) and 23 months old (black column) mice are compared side by side. The figure is based on the data of Ono et al. (2000).
4. What are the consequences of a high level of mutation Mutation theory assumes that a high level of mutation deteriorates cell as well as tissue func-
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tions. However, it has not yet been determined if this is really the case. Even if it is, the question of what level of mutation is deleterious remains to be answered. A recent development in creating KO mice could provide a clue in this regard. The several KO mouse strains deficient in one of the DNA repair genes have been shown to sustain elevated levels of mutation and/or some defects in tissue function. These strains and mutations/defects are listed in Table 2. The repair systems that have been inactivated by destruction of a gene include mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), non-homologous end joining (NHEJ), recombination and telomere maintenance. We did not include KO mice of topoisomerase IIIb (Kwan and Wang, 2001) in the Table because the role of this system in vivo is not yet clear and it could be involved in something other than DNA repair. It is noted that the topoisomerase IIIb KO mice have a short life span. Although most of the mutation studies done using the KO mice listed in Table 2 are involved young mice, the mutation frequencies were elevated in several mice. The absence of elevation of mutation in some mice can be explained easily by the possible existence of other repair pathway(s) operating on the same DNA damage. The inactivation of the three genes involved in MMR, Mlh1 , Pms2 , and Msh2 , raised mutation in many types of tissue to extremely high levels and resulted in the early occurrence of tumor in the thymus or intestine. The mutation frequencies in the brain and heart of Msh2 (///) mice have been shown to be elevated. However, the early death of the mice makes it difficult to assess whether the elevation of mutation in brain and heart accelerates aging processes in the tissues. Mutation frequency in the liver of Ogg1 -deficient mice showed about a 2-fold elevation at a young age (Klungland et al., 1999; Minowa et al., 2000). The livers of these mice also contain a high level of 8-oxoguanine in the genomic DNA. The histological studies, however, showed no lesions up to at least 1 year of age. These findings may suggest that a slight elevation of mutation can be
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tolerable for tissues in vivo. Another possibility is that some lesions could become overt later in the aging process, perhaps at older ages than 1 year. Further study is necessary to elucidate this point. Findings in a recent study on Xp c(///) mice by Wijnhoven et al. (2000), who found that the Xpc KO mice did not show any elevation of mutation at a young age but showed 30 times as many mutations in splenic T cells at 12 and 18 months of age, are of interest. In spite of the high mutation rate, the mice did not reveal any elevation of tumor incidence or accelerated aging in the tissues. Those results might indicate that a high rate of mutation is tolerable in some cases and does not result in any tissue lesion. However, it is not clear whether the elevation of mutation is limited to T-cells or is common to other tissues. Further studies on different types of tissue as well as on different kinds of NER-deficient mice will provide important clues. On the other hand, Ercc1- and Ku86 -deficient mice showed early onset of age-associated alterations in many types of tissue (Weeda et al., 1997; Vogel et al., 1999). Although mutations in these mice have not yet been studied, caution is essential in interpreting the results obtained using these mice. The alteration observed might not be directly related to the deficiency in DNA repair. For example, Ku86 is known to be involved in establishment of immunological diversity of lymphocytes. Thus, the tissue damage in adult mice could be a consequence of immuno-deficiency rather than of DNA repair-deficiency. The Ercc1-deficient mice also show a high rate of embryonic lethality, reduced body weight at birth, and lethality at about 1 month of age. The senescent phenotypes observed in many types of tissue from the mice could be a result of developmental failure induced by the Ercc1 -deficiency rather than by mutation. The reason for why development is affected by the gene destruction is unclear. The results in Blm KO mice and telomerasedeficient mice indicate that the destruction of chromosomal structure could also lead to accelerated aging (Rudolph et al., 1999; Luo et al., 2000).
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Table 2 Effects of repair gene inactivation on mutation and tissue lesion Inactivated gene
Type of repair involveda
Mutation (Marker)b (Tissues)
Mlh1
Pms2
MMR
MMR
Old r
/// (microsatellite, Ouab ) (small intestine, fibroblasts) /// (SupF, lacI) (small intestine, colon, skin) /// (micrtosatellite, Ouabr) Lymphoma (small intestine, fibroblasts) /// (SupF, lacI) (small intestine, colon, skin) /// (microsatellite, lacI) (small intestine, thymus, brain, heart)
Tissue lesion Tumor in intestine
Yao et al., 1999; Baross-Francis et al., 2001 Prolla et al., 1998
Msh2
MMR
Msh6 Ogg1
MMR BER
/ (microsatellite) (tumor) / (gpt, lacI) (liver)
Tumor in intestine, lymphoma (increase of 8-oxyguanine in DNA)
Ung Xpa
BER NER
/ (lacI) (spleen, thymus) / (rpsL) (skin)
(increase of U in DNA) Increased UV sensitivity
Xpc Ercc1
NER NER
Ku86
NHEJ
Blm mTR
Recombination Telomere maintenance
a b
/ (HPRT) (splenic T-cell)
/// (LOH at HPRT)
/ (lacZ) (liver) / (HPRT) (splenic T-cell) /// (HPRT)
Prolla et al., 1998
Thymoma
Increased UV sensitivity Delay in development and growth, lesions in many tissues Delay in growth, lesions in bone, skin and liver High incidence of various kinds of tumors Acceleration of many aging phenotypes in three to six generations in parallel with telomere shortening
MMR, mismatch repair; BER, base excision repair; NER, nucleotide excision repair; NHEJ, non-homologous end joining. /, same level as wild type; /, an increase of less than 3-fold; ///, an increase of more than 10-fold.
Yao et al., 1999; Baross-Francis et al., 2001 Wind et al., 1995; Andrew et al., 1998; Baross-Francis et al., 1998 Edelmann et al., 1997 Klungland et al., 1999; Minowa et al., 2000 Nilsen et al., 2000 Murai et al., 2000 Giese et al., 1999 Wijnhoven et al., 2000 Wijnhoven et al., 2000 Weeda et al., 1997 Vogel et al., 1999 Luo et al., 2000 Rudolph et al., 1999
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Young
References
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Determination of the exact molecular mechanisms is pending. Overall, the study of the correlations between mutation and phenotypic alteration in tissues in DNA repair gene KO mice is still in the preliminary stages. Further study will provide data that will prove or disprove the mutation theory of aging.
5. Future studies The studies reviewed above clearly indicate that mutation in the aged mouse is tissue-specific. Since mutation frequency as well as mutation spectrum seem to be similar among the tissues in newborn and young mice, the mechanism behind the tissuespecificity must become operational in the aging process after maturation. A major cause of ageassociated accumulation of mutation does not seem to be related to an error associated with DNA replication, based on the fact that non- or slowly-proliferating tissues such as that in the heart and liver show a rate of age-associated accumulation of mutation higher than that of highly proliferating tissues such as the skin and testis. What, then, might be the cause of these mutation accumulation? The cause could be tissuespecific DNA damage or DNA repair capability. At present, it is difficult to say which is more important. Possible approaches would be to elevate or reduce mutation by artificial manipulation. Recently, radiation has been proved to induce mutation in many tissues in the mouse (Ono et al., 1999; Nakamura et al., 2000). The molecular nature of mutation induced by radiation, however, appears to be different from those observed in the tissues of old mouse. This finding indicates that natural background radiation is not the major cause of mutation that accumulates in the aging process. The rate of radiation-induced mutation in the brain is about one-half that in the spleen (Nakamura et al., 2000), and corresponds to the low rate of age-related mutation accumulation in the brain when compared with that in the spleen (Table 1). It
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has previously been demonstrated that the number of DNA breaks induced by radiation as well as their repair rates are similar between proliferating and non-proliferating tissues (Ono and Okada, 1974, 1978). These data might suggest that the repair system in the brain is more accurate for some reason. Similar analyses using different kinds of chemical mutagens and reactive oxygens will provide clues for understanding the cause of the mutation accumulating in the aging process. At the same time, the possible importance of the DNA repair system should be established. We do not know whether the DNA repair system alters with age or whether it differs among various types of tissue. Expression of each DNA repair gene should be examined in individual tissues throughout the aging process. Each type of tissue might have a unique DNA protection system, as has been suggested by radiation-induced mutation studies (Nakamura et al., 2000). Among the many kinds of DNA repair genes, the thymine DNA glycosylase gene (Tdg ) would be interesting to investigate because it has been proved to be involved in a repair process responsible for the G:C to A:T mutation at the 5?CG3? sequence, which is the most frequent mutation observed in many types of tissue (Wiebauer and Jiricny, 1989). This type of mutation is assumed to be created by three steps (Gonzalgo and Jones, 1997): (i) methylation of cytosine at position 5 in the 5?CG3? sequence
Fig. 3. Schematic illustration of a formation of G:T mismatch at the 5?CG3? DNA sequence, which leads to base substitution of C:G to T:A. mC indicates 5 methylcytosine. TDG is thymine DNA glycosylase. The mutagenic T:G mismatch is repaired by TDG.
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resulting in 5-methylcytosine formation, (ii) spontaneous deamination at position 4 of 5-methylcytosine, which results in formation of T and T:G mispair instead of C:G, and (iii) replication of DNA leading to T:A and C:G pairing in the two daughter strands (Fig. 3). In mammals, the T:G mispairing could be repaired by TDG. If this is the case in tissues, an elevation of this enzyme level should reduce mutation frequency. Conversely, an inactivation of the Td g gene should raise mutation frequency. Our preliminary study on the Tdg gene KO mice, however, showed lethality of the mouse at the embryonic stage (Saito et al., 2001). Similar phenomena have been observed with destruction of the other repair genes (Friedberg and Meira, 1999). These findings indicate that some of the DNA repair proteins have a vital importance at the embryonic stage. In these cases, the significance of the protein in the aging process could be elucidated only if the responsible gene is inactivated after maturation. This inactivation can be achieved by applying the technique of conditional gene knockout (Lewandoski, 2001). Although this technique is as yet incomplete, inactivation of a specific gene in a certain tissue at a desired time will become feasible in the near future. The application of this method will assist us in separating the embryonic stage from the aging process and also in modifying genes in each type of tissue. To this end, we should be attentive to the development of new biotechnologies.
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