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Mutational fingerprints of aging

Mutational fingerprints of aging © 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30, No. 2 545–549 Martijn E. T. Dollé*, Wendy K. Snyder, David B. Dunson and Jan Vijg Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, 15355 Lambda Drive, STCBM 2.200, San Antonio, TX 78245, USA and Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA Received August 27, 2001; Revised November 7, 2001; Accepted November 15, 2001 ABSTRACT coupled to transcription might be under-represented or not detected at all in the silent reporter gene. Using a lacZ plasmid transgenic mouse model, Using the plasmid transgenic mouse model, we have previ- spectra of spontaneous point mutations were deter- ously reported organ-specific differences in mutation accumula- mined in brain, heart, liver, spleen and small intestine tion with age (4,5). Further characterization of the mutational in young and old mice. While similar at a young age, spectra as they unfold in old age could provide molecular the mutation spectra among these organs were fingerprints to obtain an insight into the possible sources of significantly different in old age. In brain and heart molecular damage, which has been implicated as the ultimate G:C→A:T transitions at CpG sites were the predomi- cause of aging and its associated diseases (6,7). Here we nant mutation, suggesting that oxidative damage is specifically compare the point mutational spectra in five organs, with different proliferative histories, in young and old not a major mutagenic event in these tissues. Other mice and in lymphomas, the most frequent neoplastic lesion in base changes, especially those affecting A:T base old age in these mice. The results indicate that the mutation pairs, positively correlated with increasing prolifera- spectra in the different organs diverge during the aging tive activity of the different tissues. A relatively high process. It is suggested that the organ-specific mutation spectra percentage of base changes at A:T base pairs and emerging in old age reflect a combination of the proliferative compound mutants were found in both spleen and history and unique function of each organ. spontaneous lymphoma, suggesting a possible role of the hypermutation process in splenocytes in MATERIALS AND METHODS carcinogenesis. The similar mutant spectra observed at a young age may reflect a common mutation mech- Plasmid rescue anism for all tissues that could be driven by the rapid Aging cohorts of male C57Bl/6 pUR288-lacZ mice of line 60 cell division that takes place during development. were maintained in the animal facilities of the Beth Israel However, the spectra of the young tissues did not Deaconess Medical Center (Boston, MA) as described previ- resemble that of the most proliferative aged tissue, ously (4). The animals were killed by decapitation following implying that replicative history per se is not the asphyxiation with CO . Organs and tissues were removed, underlying causal factor of age-related organ- rinsed in PBS, placed in 1.5 ml microcentrifuge tubes and specific differences in mutation spectra. Rather, frozen on dry ice. Any macroscopic lesions observed during differences in organ function, possibly in association tissue collection were excised and stored separately. The with replicative history, may explain the divergence tissues were maintained at –80°C until used. DNA was in mutation spectra during aging. extracted by routine phenol/chloroform extraction. Complete protocols for plasmid rescue and mutant frequency determina- tions with this model are given elsewhere (8). Briefly, between INTRODUCTION 10 and 20 µ g genomic DNA was digested with HindIII for 1 h in the presence of magnetic beads (Dynal) pre-coated with Somatic mutations are thought to play a major causal role in lacI–lacZ fusion protein. The beads were washed three times to cancer and, possibly, aging (1,2). To monitor tissue-specific patterns of somatic mutation accumulation during aging, a remove the unbound mouse genomic DNA. Plasmids were plasmid transgenic mouse model sensitive to a broad range of subsequently eluted from the beads with IPTG. After circular- mutational events has been developed (3). These mice harbor ization of the plasmids with T4 DNA ligase they were ethanol chromosomally integrated plasmids that can be efficiently precipitated and used to electrotransform E.coli C (∆ lacZ, recovered from genomic DNA and transferred into a suitable galE ) cells. One-thousandth of the transformed cells were Escherichia coli host for mutant selection, quantitation and plated on a titer plate (with X-gal) and the remainder on a characterization. The advantages of this system include an selective plate (with p-gal). The plates were incubated for 15 h extensive choice of tissue types suitable for mutation examination at 37°C. Mutant frequencies were determined as the number of and the absence of any selection pressure in vivo of a mutation colonies on the selective plates versus the number of colonies in the neutral reporter. On the other hand, mutagenic events on the titer plate (times the dilution factor of 1000). *To whom correspondence should be addressed. Tel: +1 210 562 5027; Fax: +1 210 562 5028; Email: dolle@uthscsa.edu 546 Nucleic Acids Research, 2002, Vol. 30, No. 2 Mock-recovery spleen (P = 0.03) and small intestine (P = 0.03), but not in the heart (P = 0.13) or liver (P = 0.88). In addition to these effects, To check for a possible E.coli contribution to the spontaneous the old mice exhibited clear differences between organs in both mutation spectra as observed in the lacZ plasmids obtained the mutation frequencies (P < 0.01) and subclass proportions from the mouse, the same plasmids were grown in E.coli. For (P < 0.01). However, such differences were not apparent these experiments, E.coli C cells harboring the wild-type among the younger mice (P = 0.09 and P = 0.81, respectively). pUR288 plasmid were obtained in the form of a dark blue The point mutational spectra in the five organs, which are staining colony on a titer plate from a regular mutant frequency similar at a young age, diverge in a way that, at least in part, determination. These cells were grown in 3 ml of LB medium seems to reflect their proliferative history over the lifespan of containing 75 µ g/ml ampicillin and 25 µ g/ml kanamycin for 8 h the mouse. The organs in Figure 1 have been arranged from left at 37°C at 225 r.p.m. Cells were harvested by centrifugation to right by increasing proliferative activity. Studies comparing for 10 min at 1000 g. DNA was extracted by routine phenol/ DNA-incorporated radioactivity per organ after administration chloroform extraction. The plasmid preparations were mixed of [ H]thymidine clearly set the small intestine apart from the with non-transgenic liver DNA, after which mutant plasmids other four organs in terms of proliferative activity (11,12). were recovered as described above for genomic DNA isolation. Spleen is the second most proliferative organ among the five organs studied. Both Kuppfer and parenchymal cells contributed Mutant classification to some remaining proliferative activity in the liver (11). Based Mutant colonies were taken from the selective plates and on the cardiomyocytes, the heart is virtually a post-mitotic grown overnight in 3 ml of LB medium. Then, 1 µ l was organ, but some proliferative activity, possibly due to other directly plated onto X-gal to screen for galactose-insensitive (12). Brain appears to be virtually cell types, has been found host cells (9). The remainder of the cell culture was used for devoid of proliferative activity (11,12). From our present plasmid mini preparation (Wizard 9600; Promega). The purified studies it appears that G:C→A:T transitions at CpG sites corre- plasmids were digested with PstI and AvaI and size separated late strongly with a lack of proliferative activity over a lifetime on 1% agarose gels. Mutant plasmids with restriction patterns (Fig. 1). Other base changes than G:C→A:T transitions emerge resembling and deviating from the wild-type restriction pattern with increasing frequency from brain to small intestine, the were classified as ‘no-change’ and ‘size-change’ mutants, latter organ being dramatically different, with relatively high respectively. frequencies of G:C→A:T at non-CpG sites, G:C→T:A, G:C→G:T and base changes at A:T base pairs. In general, Sequencing while G:C→A:T transitions at CpG sites are predominantly Sequencing reactions were performed with the CEQ dye found in post-mitotic organs, changes at A:T base pairs corre- terminator cycle sequencing kit (Beckman, Fullerton, CA), late positively with proliferative activity (Fig. 1). according to the manufacturer’s standard protocol, and The increase in G:C→A:T transitions at CpG sites in the analyzed with a CEQ 2000 DNA analysis system (Beckman). brain and heart indicates that the predominant mutational The primers used were as described earlier (9). mechanism in post-mitotic tissue during aging is spontaneous deamination of 5-methylcytosine. As proposed by MacPhee (13), mismatch repair would have a 50% chance of reverting a RESULTS AND DISCUSSION C:T mismatch to the original sequence and a 50% chance of Mutant lacZ plasmids were recovered from brain, heart, liver, creating a stable G:C→A:T transition in the absence of proper spleen and small intestine of young (3–4 months) and old strand recognition signals. However, most spontaneous deamin- (30–33 months) pUR288 C57Bl/6 mice and subdivided, based ations of 5-methylcytosine are repaired correctly, for instance on size, into no-change and size-change mutants. From the by specific glycosylases (14). no-change mutants, which were presumed to be point muta- Oxidative stress has been suggested to play an important tions, 20–22 were randomly selected per age/organ group from role in aging, damaging DNA and other macromolecules alike three to four animals and completely sequenced (Table 1). The (15). Because our data indicate that age-related mutation accu- point mutational spectra, limited to base changes and single mulation in the brain and heart is mainly due to spontaneous base deletions, of the five organs in the two age groups are deamination of 5-methylcytosine, oxidative damage does not expressed as mutant frequencies in Figure 1. Statistical analyses seem to be a major mutagenic event in these tissues. In this of the mutational frequencies and spectra were conducted respect, it is conceivable that the relatively high rate of oxida- using a Bayesian approach (10). In testing for differences in tive metabolism in brain and heart mainly causes mutations in the mutational spectra between groups, the mean square error the mitochondrial genome (16), while oxidative damage to the was used as the measure of discrepancy. nuclear genome of these post-mitotic tissues might be repaired The total and categorized point mutation frequencies were without a mutagenic consequence. higher on average for the older mice (a posteriori P value < 0.01). A question of major importance is the source of the muta- An increase in the frequency of all point mutations was tions in the more proliferative organs, most notably the small observed in the heart (P < 0.01), liver (P < 0.01), spleen (P = 0.01) intestine, which are likely to be caused by misreplication at and small intestine (P < 0.01), but not in the brain (P = 0.41). damaged sites. In this respect, at least two possibilities come to The magnitude of the difference between young and old mice mind. First, oxidative damage might play a role, since predomi- was higher in the small intestine than in the other organs (P = 0.05). nantly G:C base pairs were affected (Fig. 1). G or C bases have There were also clear differences between age groups in the been identified as the main target for oxidative damage in vitro proportions of mutations falling into the different subclasses (17). Second, replication errors may also arise as a conse- (P < 0.01). This difference was evident in the brain (P < 0.01), quence of DNA lesions induced by environmental mutagens, Nucleic Acids Research, 2002, Vol. 30, No. 2 547 Table 1. Sequenced lacZ no-change mutations recovered from brain, liver, spleen and lymphoma of 3.5- and 32-month-old mice Empty lines separate mutants obtained from individual mice. The cross-hatched areas indicate compound mutants; all other mutants contained single mutations. Sequence data on point mutations for heart and small intestine have been published elsewhere (5). Nucleotide numbering according to SYNPUR288V (GenBank accession no. L09147). Frequency of recurrent mutations, i.e. identical mutants recovered from the same tissue sample. Other seemingly recurrent mutations in this table were unique, based on the presence of different polymorphic markers among the mutated plasmids. (These single nucleotide polymorphisms were shown to be present among the integrated wild-type plasmid copies of this transgenic mouse model; 26.) G:C→A:T base change at a CpG site. taken up with food by the small intestine or detoxified in the mutants sequenced contained multiple mutations, i.e. 14%. In liver. spleen, the main target organ for lymphomas in aging mice, the frequency of lacZ mutants containing two or more mutations In old spleen a relatively high percentage of base changes was 10%, while averaging only 2% in the other four organs was found to involve A:T base pairs (Fig. 1). This is in keeping (Table 1). We interpret the high percentage of such compound with the observed point mutational spectra of both human and mouse lymphocytes at the Hprt locus (18,19). In this respect, it mutants as evidence for a temporal burst of mutational activity appears that Hprt mutation spectra in blood lymphocytes are at some point in the history of these tissues. As such, this finding is in keeping with the mutator phenotype postulated to not representative of other cell and tissue types. Interestingly, a underlie the initiation and progression of tumors (20). It is high frequency of base changes at A:T was also found in the mutation spectrum of spontaneous lymphomas isolated from tempting to speculate that somatic hypermutation, a predomi- old mice (Fig. 2). Two of the fourteen unique lymphoma nantly point mutational process improving the affinity of Ig 548 Nucleic Acids Research, 2002, Vol. 30, No. 2 Figure 1. Point mutational spectra of the lacZ reporter gene in brain, heart, liver, spleen and small intestine from young (3.5 months) and old (32 months) mice. Bars represent the frequency of each type of point mutation as indicated. The black areas in the G:C→A:T bars indicate the fraction of such mutations that had occurred at CpG sites and the gray areas in the Del (–1) bars indicate the fraction of such mutations that had occurred at reiterated sites, i.e. a sequence of three or more of the same nucleotide. Corrections for recurrent mutations were made. not apply to the small intestine. Indeed, while small intestine has the highest spontaneous mutation rate of all tissues tested (Fig. 1) (5), tumors in this tissue occur at very low frequencies (23). Possibly, other factors than somatic mutation rate alone play a role in determining susceptibility of an organ to tumor formation. In this respect, it should be noted that mice deficient for the mismatch repair genes Mlh1 and Pms2 show 18- and 13-fold increases in point mutations in the small intestine at a lacI transgene (24), respectively, which did not dramatically increase the frequency of small intestinal tumors (25). At a young age the mutation spectra in brain, heart, liver, spleen and small intestine are remarkably similar (Fig. 1). To exclude the possibility that this similarity is due to a back- ground level of mutations due to the rescue process, a point mutational spectrum from mock-recovered plasmids grown in E.coli was determined (Fig. 3). Although the mock-recovered Figure 2. Point mutational spectra of the lacZ reporter gene in spontaneous spectrum resembled the spectra of the young mouse tissues lymphomas found in old mice. See legend to Figure 1 for an explanation of the (Figs 1 and 3), the mock-recovered point mutant frequency was bars. Mutational spectra are expressed as percentages to omit inaccuracies in –5 –5 only ∼0.6 × 10 , as compared with 2.8 × 10 on average in correcting mutant frequencies for frequent recurrent mutations leading to a tissues from young animals. Furthermore, the mock-recovered small number of unique plasmids analyzed for some lymphomas (Table 1). mutant frequency is an overestimate due to the inability to obtain mutation-free starting material, i.e. wild-type plasmid molecules in germinal centers in lymph nodes and spleen, preparations. Based on our previous results (26), many of the causes such compound mutants, which may increase cancer mutations occur during growth in E.coli prior to plasmid risk. Recently, somatic hypermutation has been associated with DNA polymerase η as an A:T mutator (21,22), which preparation and not during mock recovery. Indeed, when the could explain the relatively large fraction of mutations found at sites of the base changes were taken into account, many of the A:T base pairs in old spleen and lymphoma (Figs 1 and 2). mock-recovered mutants turned out to be unique, i.e. only 17% While the observed mutation spectrum in the spleen could be of the point mutations were found among 140 different point causally related to the etiology of lymphomas, the same does mutations recovered from the mouse. The young mouse tissues Nucleic Acids Research, 2002, Vol. 30, No. 2 549 REFERENCES 1. Vijg,J. (2000) Somatic mutations and aging: a re-evaluation. Mutat. Res., 447, 117–135. 2. DePinho,R.A. (2000) The age of cancer. Nature, 408, 248–254. 3. Boerrigter,M.E.T.I., Dollé,M.E.T., Martus,H.-J., Gossen,J.A. and Vijg,J. (1995) Plasmid-based transgenic mouse model for studying in vivo mutations. Nature, 377, 657–659. 4. Dollé,M.E.T., Giese,H., Hopkins,C.L., Martus,H.-J., Hausdorff,J.M. and Vijg,J. (1997) Rapid accumulation of genome rearrangements in liver but not in brain of old mice. Nature Genet., 17, 431–434. 5. Dollé,M.E.T., Snyder,W.K., Gossen,J.A., Lohman,P.H. and Vijg,J. (2000) Distinct spectra of somatic mutations accumulated with age in mouse heart and small intestine. Proc. Natl Acad. Sci. USA, 97, 8403–8408. 6. Kirkwood,T.B. and Austad,S.N. (2000) Why do we age? Nature, 408, 233–238. 7. Vijg,J. and Dollé,M.E.T. (2001) Instability of the nuclear genome and the role of DNA repair. In Masoro,E.J. and Austad,S.N. (eds), Handbook of the Biology of Aging, 5th Edn. Academic Press, San Diego, CA, pp. 84–113. 8. Vijg,J., Boerrigter,M.E.T.I. and Dollé,M.E.T. (1999) A transgenic mouse Figure 3. Point mutational spectra of mock-recovered plasmids grown in E.coli C. model for studying mutations in vivo. In Yu,B.P. (ed.), Methods in Aging See legend to Figure 1 for an explanation of the bars. Mutational spectra are Research: Section D. CRC Press, Boca Raton, FL, pp. 621–635. expressed as percentages, since thus far there is no evidence that they represent a 9. Dollé,M.E.T., Martus,H.-J., Novak,M., van Orsouw,N.J. and Vijg,J. real background, i.e. find their origin in the rescue process (see text). (1999) Characterization of color mutants in lacZ plasmid-based transgenic mice, as detected by positive selection. Mutagenesis, 14, 287–293. 10. Dunson,D.B. and Tindall,K.R. (2000) Bayesian analysis of mutational Table 2. Number of identical point mutations found in the lacZ transgene of spectra. Genetics, 156, 1411–1418. other mouse tissues 11. Edwards,J.L. and Klein,R.E. (1961) Cell renewal in adult mouse tissues. Am. J. Pathol., 38, 437–453. 12. Hinrichs,H.R., Petersen,R.O. and Baserga,R. (1964) Incorporation of Source Compared Matching Percentage thymidine into DNA of mouse organs. Arch. Pathol., 78, 245–253. Young brain 20 5 25 13. MacPhee,D.G. (1995) Mismatch repair, somatic mutations and the origins Young heart 20 10 50 of cancer. Cancer Res., 55, 5489–5492. Young liver 20 11 55 14. Hendrich,B., Hardeland,U., Ng,H.-H., Jiricny,J. and Bird,A. (1999) The thymine glycosylase MBD4 can bind to the product of deamination at Young spleen 23 8 35 methylated CpG sites. Nature, 401, 301–304. Young small intestine 19 6 32 15. Sohal,R.S. and Weindruch,R. (1996) Oxidative stress, caloric restriction Average young mouse tissues 39 and aging. Science, 273, 59–63. 16. Lee,C.M., Weindruch,R. and Aiken,J.M. (1997) Age-associated alterations of Mock-recovery (E.coli) 18 3 17 the mitochondrial genome. Free Radic. Biol. Med., 22, 1259–1269. 17. Reid,T.M., Feig,D.I. and Loeb,L.A. (1994) Mutagenesis by metal-induced The mouse database comprised 140 unique point mutations. oxygen radicals. Environ. Health Perspect., 102 (suppl. 3), 57–61. 18. Cariello,N.F., Douglas,G.R. and Soussi,T. (1996) Databases and software for the analysis of mutations in the human p53 gene, the human hprt gene shared on average 39% of their point mutations with this and the lacZ gene in transgenic rodents. Nucleic Acids Res., 24, 119–120. mouse mutation database (Table 2). 19. Meng,Q., Singh,N., Heflich,R.H., Bauer,M.J. and Walker,V.E. (2000) Comparison of the mutations at Hprt exon 3 of T-lymphocytes from Hence, we believe that the mutation spectra in the young B6C3F1 mice and F344 rats exposed by inhalation to 1,3-butadiene or the somatic tissues (Fig. 1) are genuinely similar, which suggests racemic mixture of 1,2:3,4-diepoxybutane. Mutat. Res. 464, 169–184. that, in contrast to aging, development is associated with a 20. Loeb,L.A. (2001) A mutator phenotype in cancer. Cancer Res., 61, 3230–3239. mutation mechanism common to all cells and tissues. In this 21. Rogozin,I.B., Pavlov,Y.I., Bebenek,K., Matsuda,T. and Kunkel,T.A. respect, one would suspect that these early mutation spectra are (2001) Somatic mutation hotspots correlate with DNA polymerase eta associated with replication errors and, hence, resemble the error spectrum. Nature Immunol., 2, 530–536. 22. Zeng,X., Winter,D.B., Kasmer,C., Kraemer,K.H., Lehmann,A.R. and spectra in actively proliferating tissue, such as spleen and small Gearhart,P.J. (2001) DNA polymerase η is an A-T mutator in somatic intestine, at a greater age. However, this is not the case, which hypermutation of immunoglobulin variable genes. Nature Immunol., 2, suggests that replicative history per se is not the underlying 537–541. causal factor of age-related organ-specific mutation spectra. 23. Bult,C.J., Krupke,D.M., Naf,D., Sundberg,J.P. and Eppig,J.T. (2001) Web-based access to mouse models of human cancers: the Mouse Tumor Rather, differences in organ function, possibly in association Biology (MTB) Database. Nucleic Acids Res., 29, 95–97. with replicative history, may explain the divergence in muta- 24. Baross-Francis,A., Makhani,N., Liskay,R.M. and Jirik,F.R. (2001) tion spectra during aging. Such in vivo mutational fingerprints Elevated mutant frequencies and increased C:G→T:A transitions in are likely to provide clues as to the various sources of somatic Mlh1–/– versus Pms2–/– murine small intestinal epithelial cells. Oncogene, 20, 619–625. damage thought to underlie age-related cellular degeneration 25. Prolla,T.A., Baker,S.M., Harris,A.C., Tsao,J.L., Yao,X., Bronner,C.E., and death under various environmental conditions. Zheng,B., Gordon,M., Reneker,J., Arnheim,N., Shibata,D., Bradley,A. and Liskay,R.M. (1998) Tumour susceptibility and spontaneous mutation in mice deficient in Mlh1, Pms1 and Pms2 DNA mismatch repair. Nature ACKNOWLEDGEMENTS Genet., 18, 276–279. 26. Dollé,M.E.T., Snyder,W.K., van Orsouw,N.J. and Vijg,J. (1999) This work was supported by grants 1PO1AG17242-01, Background mutations and polymorphisms in lacZ-plasmid transgenic AG13319-05 and 1RO1CA75653-03. mice. Environ. Mol. Mutagen., 34, 112–120. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Mutational fingerprints of aging

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© 2002 Oxford University Press Nucleic Acids Research, 2002, Vol. 30, No. 2 545–549 Martijn E. T. Dollé*, Wendy K. Snyder, David B. Dunson and Jan Vijg Sam and Ann Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center, 15355 Lambda Drive, STCBM 2.200, San Antonio, TX 78245, USA and Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA Received August 27, 2001; Revised November 7, 2001; Accepted November 15, 2001 ABSTRACT coupled to transcription might be under-represented or not detected at all in the silent reporter gene. Using a lacZ plasmid transgenic mouse model, Using the plasmid transgenic mouse model, we have previ- spectra of spontaneous point mutations were deter- ously reported organ-specific differences in mutation accumula- mined in brain, heart, liver, spleen and small intestine tion with age (4,5). Further characterization of the mutational in young and old mice. While similar at a young age, spectra as they unfold in old age could provide molecular the mutation spectra among these organs were fingerprints to obtain an insight into the possible sources of significantly different in old age. In brain and heart molecular damage, which has been implicated as the ultimate G:C→A:T transitions at CpG sites were the predomi- cause of aging and its associated diseases (6,7). Here we nant mutation, suggesting that oxidative damage is specifically compare the point mutational spectra in five organs, with different proliferative histories, in young and old not a major mutagenic event in these tissues. Other mice and in lymphomas, the most frequent neoplastic lesion in base changes, especially those affecting A:T base old age in these mice. The results indicate that the mutation pairs, positively correlated with increasing prolifera- spectra in the different organs diverge during the aging tive activity of the different tissues. A relatively high process. It is suggested that the organ-specific mutation spectra percentage of base changes at A:T base pairs and emerging in old age reflect a combination of the proliferative compound mutants were found in both spleen and history and unique function of each organ. spontaneous lymphoma, suggesting a possible role of the hypermutation process in splenocytes in MATERIALS AND METHODS carcinogenesis. The similar mutant spectra observed at a young age may reflect a common mutation mech- Plasmid rescue anism for all tissues that could be driven by the rapid Aging cohorts of male C57Bl/6 pUR288-lacZ mice of line 60 cell division that takes place during development. were maintained in the animal facilities of the Beth Israel However, the spectra of the young tissues did not Deaconess Medical Center (Boston, MA) as described previ- resemble that of the most proliferative aged tissue, ously (4). The animals were killed by decapitation following implying that replicative history per se is not the asphyxiation with CO . Organs and tissues were removed, underlying causal factor of age-related organ- rinsed in PBS, placed in 1.5 ml microcentrifuge tubes and specific differences in mutation spectra. Rather, frozen on dry ice. Any macroscopic lesions observed during differences in organ function, possibly in association tissue collection were excised and stored separately. The with replicative history, may explain the divergence tissues were maintained at –80°C until used. DNA was in mutation spectra during aging. extracted by routine phenol/chloroform extraction. Complete protocols for plasmid rescue and mutant frequency determina- tions with this model are given elsewhere (8). Briefly, between INTRODUCTION 10 and 20 µ g genomic DNA was digested with HindIII for 1 h in the presence of magnetic beads (Dynal) pre-coated with Somatic mutations are thought to play a major causal role in lacI–lacZ fusion protein. The beads were washed three times to cancer and, possibly, aging (1,2). To monitor tissue-specific patterns of somatic mutation accumulation during aging, a remove the unbound mouse genomic DNA. Plasmids were plasmid transgenic mouse model sensitive to a broad range of subsequently eluted from the beads with IPTG. After circular- mutational events has been developed (3). These mice harbor ization of the plasmids with T4 DNA ligase they were ethanol chromosomally integrated plasmids that can be efficiently precipitated and used to electrotransform E.coli C (∆ lacZ, recovered from genomic DNA and transferred into a suitable galE ) cells. One-thousandth of the transformed cells were Escherichia coli host for mutant selection, quantitation and plated on a titer plate (with X-gal) and the remainder on a characterization. The advantages of this system include an selective plate (with p-gal). The plates were incubated for 15 h extensive choice of tissue types suitable for mutation examination at 37°C. Mutant frequencies were determined as the number of and the absence of any selection pressure in vivo of a mutation colonies on the selective plates versus the number of colonies in the neutral reporter. On the other hand, mutagenic events on the titer plate (times the dilution factor of 1000). *To whom correspondence should be addressed. Tel: +1 210 562 5027; Fax: +1 210 562 5028; Email: dolle@uthscsa.edu 546 Nucleic Acids Research, 2002, Vol. 30, No. 2 Mock-recovery spleen (P = 0.03) and small intestine (P = 0.03), but not in the heart (P = 0.13) or liver (P = 0.88). In addition to these effects, To check for a possible E.coli contribution to the spontaneous the old mice exhibited clear differences between organs in both mutation spectra as observed in the lacZ plasmids obtained the mutation frequencies (P < 0.01) and subclass proportions from the mouse, the same plasmids were grown in E.coli. For (P < 0.01). However, such differences were not apparent these experiments, E.coli C cells harboring the wild-type among the younger mice (P = 0.09 and P = 0.81, respectively). pUR288 plasmid were obtained in the form of a dark blue The point mutational spectra in the five organs, which are staining colony on a titer plate from a regular mutant frequency similar at a young age, diverge in a way that, at least in part, determination. These cells were grown in 3 ml of LB medium seems to reflect their proliferative history over the lifespan of containing 75 µ g/ml ampicillin and 25 µ g/ml kanamycin for 8 h the mouse. The organs in Figure 1 have been arranged from left at 37°C at 225 r.p.m. Cells were harvested by centrifugation to right by increasing proliferative activity. Studies comparing for 10 min at 1000 g. DNA was extracted by routine phenol/ DNA-incorporated radioactivity per organ after administration chloroform extraction. The plasmid preparations were mixed of [ H]thymidine clearly set the small intestine apart from the with non-transgenic liver DNA, after which mutant plasmids other four organs in terms of proliferative activity (11,12). were recovered as described above for genomic DNA isolation. Spleen is the second most proliferative organ among the five organs studied. Both Kuppfer and parenchymal cells contributed Mutant classification to some remaining proliferative activity in the liver (11). Based Mutant colonies were taken from the selective plates and on the cardiomyocytes, the heart is virtually a post-mitotic grown overnight in 3 ml of LB medium. Then, 1 µ l was organ, but some proliferative activity, possibly due to other directly plated onto X-gal to screen for galactose-insensitive (12). Brain appears to be virtually cell types, has been found host cells (9). The remainder of the cell culture was used for devoid of proliferative activity (11,12). From our present plasmid mini preparation (Wizard 9600; Promega). The purified studies it appears that G:C→A:T transitions at CpG sites corre- plasmids were digested with PstI and AvaI and size separated late strongly with a lack of proliferative activity over a lifetime on 1% agarose gels. Mutant plasmids with restriction patterns (Fig. 1). Other base changes than G:C→A:T transitions emerge resembling and deviating from the wild-type restriction pattern with increasing frequency from brain to small intestine, the were classified as ‘no-change’ and ‘size-change’ mutants, latter organ being dramatically different, with relatively high respectively. frequencies of G:C→A:T at non-CpG sites, G:C→T:A, G:C→G:T and base changes at A:T base pairs. In general, Sequencing while G:C→A:T transitions at CpG sites are predominantly Sequencing reactions were performed with the CEQ dye found in post-mitotic organs, changes at A:T base pairs corre- terminator cycle sequencing kit (Beckman, Fullerton, CA), late positively with proliferative activity (Fig. 1). according to the manufacturer’s standard protocol, and The increase in G:C→A:T transitions at CpG sites in the analyzed with a CEQ 2000 DNA analysis system (Beckman). brain and heart indicates that the predominant mutational The primers used were as described earlier (9). mechanism in post-mitotic tissue during aging is spontaneous deamination of 5-methylcytosine. As proposed by MacPhee (13), mismatch repair would have a 50% chance of reverting a RESULTS AND DISCUSSION C:T mismatch to the original sequence and a 50% chance of Mutant lacZ plasmids were recovered from brain, heart, liver, creating a stable G:C→A:T transition in the absence of proper spleen and small intestine of young (3–4 months) and old strand recognition signals. However, most spontaneous deamin- (30–33 months) pUR288 C57Bl/6 mice and subdivided, based ations of 5-methylcytosine are repaired correctly, for instance on size, into no-change and size-change mutants. From the by specific glycosylases (14). no-change mutants, which were presumed to be point muta- Oxidative stress has been suggested to play an important tions, 20–22 were randomly selected per age/organ group from role in aging, damaging DNA and other macromolecules alike three to four animals and completely sequenced (Table 1). The (15). Because our data indicate that age-related mutation accu- point mutational spectra, limited to base changes and single mulation in the brain and heart is mainly due to spontaneous base deletions, of the five organs in the two age groups are deamination of 5-methylcytosine, oxidative damage does not expressed as mutant frequencies in Figure 1. Statistical analyses seem to be a major mutagenic event in these tissues. In this of the mutational frequencies and spectra were conducted respect, it is conceivable that the relatively high rate of oxida- using a Bayesian approach (10). In testing for differences in tive metabolism in brain and heart mainly causes mutations in the mutational spectra between groups, the mean square error the mitochondrial genome (16), while oxidative damage to the was used as the measure of discrepancy. nuclear genome of these post-mitotic tissues might be repaired The total and categorized point mutation frequencies were without a mutagenic consequence. higher on average for the older mice (a posteriori P value < 0.01). A question of major importance is the source of the muta- An increase in the frequency of all point mutations was tions in the more proliferative organs, most notably the small observed in the heart (P < 0.01), liver (P < 0.01), spleen (P = 0.01) intestine, which are likely to be caused by misreplication at and small intestine (P < 0.01), but not in the brain (P = 0.41). damaged sites. In this respect, at least two possibilities come to The magnitude of the difference between young and old mice mind. First, oxidative damage might play a role, since predomi- was higher in the small intestine than in the other organs (P = 0.05). nantly G:C base pairs were affected (Fig. 1). G or C bases have There were also clear differences between age groups in the been identified as the main target for oxidative damage in vitro proportions of mutations falling into the different subclasses (17). Second, replication errors may also arise as a conse- (P < 0.01). This difference was evident in the brain (P < 0.01), quence of DNA lesions induced by environmental mutagens, Nucleic Acids Research, 2002, Vol. 30, No. 2 547 Table 1. Sequenced lacZ no-change mutations recovered from brain, liver, spleen and lymphoma of 3.5- and 32-month-old mice Empty lines separate mutants obtained from individual mice. The cross-hatched areas indicate compound mutants; all other mutants contained single mutations. Sequence data on point mutations for heart and small intestine have been published elsewhere (5). Nucleotide numbering according to SYNPUR288V (GenBank accession no. L09147). Frequency of recurrent mutations, i.e. identical mutants recovered from the same tissue sample. Other seemingly recurrent mutations in this table were unique, based on the presence of different polymorphic markers among the mutated plasmids. (These single nucleotide polymorphisms were shown to be present among the integrated wild-type plasmid copies of this transgenic mouse model; 26.) G:C→A:T base change at a CpG site. taken up with food by the small intestine or detoxified in the mutants sequenced contained multiple mutations, i.e. 14%. In liver. spleen, the main target organ for lymphomas in aging mice, the frequency of lacZ mutants containing two or more mutations In old spleen a relatively high percentage of base changes was 10%, while averaging only 2% in the other four organs was found to involve A:T base pairs (Fig. 1). This is in keeping (Table 1). We interpret the high percentage of such compound with the observed point mutational spectra of both human and mouse lymphocytes at the Hprt locus (18,19). In this respect, it mutants as evidence for a temporal burst of mutational activity appears that Hprt mutation spectra in blood lymphocytes are at some point in the history of these tissues. As such, this finding is in keeping with the mutator phenotype postulated to not representative of other cell and tissue types. Interestingly, a underlie the initiation and progression of tumors (20). It is high frequency of base changes at A:T was also found in the mutation spectrum of spontaneous lymphomas isolated from tempting to speculate that somatic hypermutation, a predomi- old mice (Fig. 2). Two of the fourteen unique lymphoma nantly point mutational process improving the affinity of Ig 548 Nucleic Acids Research, 2002, Vol. 30, No. 2 Figure 1. Point mutational spectra of the lacZ reporter gene in brain, heart, liver, spleen and small intestine from young (3.5 months) and old (32 months) mice. Bars represent the frequency of each type of point mutation as indicated. The black areas in the G:C→A:T bars indicate the fraction of such mutations that had occurred at CpG sites and the gray areas in the Del (–1) bars indicate the fraction of such mutations that had occurred at reiterated sites, i.e. a sequence of three or more of the same nucleotide. Corrections for recurrent mutations were made. not apply to the small intestine. Indeed, while small intestine has the highest spontaneous mutation rate of all tissues tested (Fig. 1) (5), tumors in this tissue occur at very low frequencies (23). Possibly, other factors than somatic mutation rate alone play a role in determining susceptibility of an organ to tumor formation. In this respect, it should be noted that mice deficient for the mismatch repair genes Mlh1 and Pms2 show 18- and 13-fold increases in point mutations in the small intestine at a lacI transgene (24), respectively, which did not dramatically increase the frequency of small intestinal tumors (25). At a young age the mutation spectra in brain, heart, liver, spleen and small intestine are remarkably similar (Fig. 1). To exclude the possibility that this similarity is due to a back- ground level of mutations due to the rescue process, a point mutational spectrum from mock-recovered plasmids grown in E.coli was determined (Fig. 3). Although the mock-recovered Figure 2. Point mutational spectra of the lacZ reporter gene in spontaneous spectrum resembled the spectra of the young mouse tissues lymphomas found in old mice. See legend to Figure 1 for an explanation of the (Figs 1 and 3), the mock-recovered point mutant frequency was bars. Mutational spectra are expressed as percentages to omit inaccuracies in –5 –5 only ∼0.6 × 10 , as compared with 2.8 × 10 on average in correcting mutant frequencies for frequent recurrent mutations leading to a tissues from young animals. Furthermore, the mock-recovered small number of unique plasmids analyzed for some lymphomas (Table 1). mutant frequency is an overestimate due to the inability to obtain mutation-free starting material, i.e. wild-type plasmid molecules in germinal centers in lymph nodes and spleen, preparations. Based on our previous results (26), many of the causes such compound mutants, which may increase cancer mutations occur during growth in E.coli prior to plasmid risk. Recently, somatic hypermutation has been associated with DNA polymerase η as an A:T mutator (21,22), which preparation and not during mock recovery. Indeed, when the could explain the relatively large fraction of mutations found at sites of the base changes were taken into account, many of the A:T base pairs in old spleen and lymphoma (Figs 1 and 2). mock-recovered mutants turned out to be unique, i.e. only 17% While the observed mutation spectrum in the spleen could be of the point mutations were found among 140 different point causally related to the etiology of lymphomas, the same does mutations recovered from the mouse. The young mouse tissues Nucleic Acids Research, 2002, Vol. 30, No. 2 549 REFERENCES 1. Vijg,J. (2000) Somatic mutations and aging: a re-evaluation. Mutat. Res., 447, 117–135. 2. DePinho,R.A. (2000) The age of cancer. Nature, 408, 248–254. 3. Boerrigter,M.E.T.I., Dollé,M.E.T., Martus,H.-J., Gossen,J.A. and Vijg,J. (1995) Plasmid-based transgenic mouse model for studying in vivo mutations. Nature, 377, 657–659. 4. Dollé,M.E.T., Giese,H., Hopkins,C.L., Martus,H.-J., Hausdorff,J.M. and Vijg,J. 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Nucleic Acids ResearchOxford University Press

Published: Jan 15, 2002

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