Hironobu Ikehata

Age-associated increase of spontaneous mutant frequency and molecular nature of mutation in newborn and old lacZ-transgenic mouse

Accumulation of mutation has long been hypothesized to be a cause of aging and contribute to many of the degenerative diseases, which appear in the senescent phase of life. To test this hypothesis, age-associated changes in spontaneous mutation in different tissues of the body as well as the molecular nature of such changes should be examined. This kind of approach has become feasible only lately with a development of new transgenic mice suitable for mutation assay. Here, using one of these transgenic mice harboring lacZ gene, we have shown that the age-associated increase in spontaneous mutant frequency is common to all tissues examined; spleen, liver, heart, brain, skin and testis, while the rates of increase in mutant frequency differed among the tissues. DNA sequencing of the 496 lacZ mutants recovered from the tissues of newborn and old mice has revealed that spectra of mutations are similar at the two age points with G:C to A:T transition at CpG site being a predominant type of mutation. Furthermore, some mutations in old tissues are complex type and not found in tissues of newborn mice. These results suggest that similar mechanisms may be operating for mutation induction in fetal and postnatal aging process. In addition, the appearance of complex types of mutations in the old tissues suggests a unique cause for these mutations in aging tissues.

A PHOSPHATIDYLINOSITOL 3-KINASE INHIBITOR WORTMANNIN INDUCES RADIORESISTANT DNA SYNTHESIS AND SENSITIZES CELLS TO BLEOMYCIN AND IONIZING RADIATION

ATM and DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) have been shown to have sequences homologous to the catalytic domains of mammalian phosphatidylinositol 3‐kinase (PI3‐kinase). In order to determine the contribution of ATM and DNA‐PKcs to the increased sensitivity of cells to DNA‐damaging agents observed in the presence of PI3‐kinase inhibitors, we examined the effects of a PI3‐kinase inhibitor, wortmannin, on cellular sensitivity to bleomycin (BLM), mitomycin C (MMC), X‐irradiation and ultraviolet (UV)‐irradiation using 2 human tumor cell lines (T98G and A172), a human fibroblast cell line (LM217), an ataxia telangiectasia (AT) cell line (AT3BISV), a scid murine cell line (SCF) and a control murine cell line (CBF). Wortmannin sensitized all of the cells, including AT3BISV and SCF, to BLM and X‐irradiation, but not to MMC or UV‐irradiation. Hypersensitivity to BLM and X‐irradiation and normal sensitivity to MMC and UV‐irradiation are characteristic phenotypes of both AT and scid mice. DNA‐dependent protein kinase (DNA‐PK) activity was suppressed by wortmannin to 45–65% of the control values in all of the cells except SCF, in which DNA‐PK activity was not detected. Wortmannin also induced radioresistant DNA synthesis, which is a cellular phenotype of AT, in T98G and SCF cells, but did not change the DNA synthesis rates after X‐irradiation in AT3BISV. Our data suggest that wortmannin decreases the activities of both the ATM protein and DNA‐PK, indicating that it might be of use as a sensitizing agent for radiotherapy and chemotherapy. Int. J. Cancer 78:642–647, 1998.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic mouse increases with age.

Spontaneous mutant frequency of lacZ gene in spleen of transgenic MutaTM mouse was examined at different ages. It was (3.2 +/- 1.3 (SD)) x 10(-5) at newborn and increased almost linearly with age up to (8.3 +/- 1.8) x 10(-5) at one year. Since the mutation of the gene is not likely to be subject to selection in vivo, the data support the idea that spontaneous mutation takes place throughout aging process and accumulates with age if not selected out by cell death.

UVB-induced gpt mutations in the skin of gpt delta transgenic mice.

Ultraviolet light B (UVB)‐induced mutagenesis was studied in gpt delta transgenic mice, which contain the λEG10 shuttle vector as a transgene. The mice were exposed to UVB at single doses of 0.3, 0.5, 1.0, 1.5, and 2.0 kJ/m2. At 4 weeks after irradiation, the mutant frequencies (MF) of the gpt gene were determined in the epidermis and the dermis, and the gpt mutations in the epidermis were identified by DNA sequencing. The epidermis exhibited a higher sensitivity to UVB than the dermis at doses of 0.3 and 0.5 kJ/m2 UVB: the MF of the epidermis were more than nine times higher than those of the nonirradiated mice, whereas the MF of the dermis were only two to three times higher than the nonirradiated level at the doses used. The UVB‐induced mutation spectrum in the epidermis was dominated by G:C to A:T transitions at dipyrimidine sites, such as 5′‐TC‐3′, 5′‐CC‐3′, and 5′‐T/C‐CG‐3′. Tandem transitions such as CC to TT were also observed. Interestingly, a remarkable bias towards the template strand of the gpt gene was observed in the single transitions at 5′‐TC‐3′ and 5′‐CC‐3′ sites, but not at 5′‐T/C‐CG‐3′ site. In contrast, G:C to A:T transitions at CpG sites and deletions were observed in nonirradiated mice. Hot spots of transitions were observed at different sites in UVB‐irradiated and nonirradiated mice. These results indicate that gpt delta transgenic mouse is a suitable model for studying in vivo UVB‐induced mutations at the molecular level. Environ. Mol. Mutagen. 34:72–79, 1999

Mutation theory of aging, assessed in transgenic mice and knockout mice

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.

Significance of CpG methylation for solar UV-induced mutagenesis and carcinogenesis in skin.

Mutations detected in the p53 gene in human nonmelanoma skin cancers show a highly UV‐specific mutation pattern, a dominance of C→T base substitutions at dipyrimidine sites plus frequent CC→TT tandem substitutions, indicating a major involvement of solar UV in the skin carcinogenesis. These mutations also have another important characteristic of frequent occurrences at CpG dinucleotide sites, some of which actually show prominent hotspots in the p53 gene. Although mammalian solar UV‐induced mutation spectra were studied intensively in the aprt gene using rodent cultured cells and the UV‐specific mutation pattern was confirmed, the second characteristic of the p53 mutations in human skin cancers had not been reproduced. However, studies with transgenic mouse systems developed thereafter for mutation research, which harbor methyl CpG‐abundant transgenes as mutation markers, yielded complete reproductions of the situation of the human skin cancer mutations in terms of both the UV‐specific pattern and the frequent occurrence at CpG sites. In this review, we evaluate the significance of the CpG methylation for solar UV mutagenesis in the mammalian genome, which would lead to skin carcinogenesis. We propose that the UV‐specific mutations at methylated CpG sites, C→T transitions at methyl CpG‐associated dipyrimidine sites, are a solar UV‐specific mutation signature, and have estimated the wavelength range effective for the solar–UV‐specific mutation as 310–340 nm. We also recommend the use of methyl CpG‐enriched sequences as mutational targets for studies on solar‐UV genotoxicity for human, rather than conventional mammalian mutational marker genes such as the aprt and hprt genes.

Spontaneous mutations in digestive tract of old mice show tissue-specific patterns of genomic instability.

In an attempt to evaluate the possible role of mutations in the age-dependent increase of tumor incidence, we studied the mutational burden that accumulates in the aging process in different parts of the digestive tract in mice. The mutations were monitored in lacZ genes integrated in the mouse genome. The digestive tract was divided into the esophagus, stomach, proximal, medial, and distal part of the small intestine, and the colon. Epithelial tissues were separated from these tissues with the exception of the esophagus, in which case the whole tissue was examined. At a young age, the mutant frequencies as well as the molecular nature of the mutations were similar among the tissues examined. In old age, on the other hand, mutant frequencies were elevated to different degrees among the tissues; they were high in the small intestine and colon, intermediate in the stomach, and low in the esophagus. The molecular characteristics of the mutations also revealed distinct tissue-specificity; there were elevated rates of a small deletion mutation in the esophagus, G:C to T:A transversion in the proximal small intestine, and multiple mutations in the distal small intestine and colon. The results indicate that different parts of the digestive tract suffer from different kinds of mutational stress in the aging process. The nature of the multiple mutations suggests the presence of a mutator phenotype based on an imbalance in deoxyribonucleotide pools.

Distribution of spontaneous CpG-associated G:C → A:T mutations in the lacZ gene of muta mice: Effects of CpG methylation, the sequence context of CpG sites, and severity of mutations on the activity of the lacZ gene product

In our previous study using transgenic Muta™ mice, G:C → A:T transitions at 5′‐CG‐3′ (CpG) sites, which are the most common mammalian spontaneous mutation, were detected in 197 of 330 spontaneous lacZ mutants. These transitions were recovered at only 27 of the 357 mutable G:C pairs within CpG sites where the transition could produce a missense or termination codon in the lacZ gene. To address the underlying mechanism for the uneven distribution of mutated CpG sites, the CpG methylation status of the Muta lacZ gene was analyzed by a bisulfite method. All the CpG sites examined in the coding region were evenly methylated at a high level, and no site‐specific methylation was evident. Analysis of the sequence context around the mutated CpG sites, however, revealed that 21 of these 27 sites contained a CpG flanked by a pyrimidine on the 5′ side, and that 187 of the 197 mutants resulted from substitutions at these sites. Moreover, we found five hotspots among those sites, the location of which was intimately related to the enzymatic activity of the gene product: one site produced a nonsense codon; three sites, one of which corresponded to the nucleophile at the active site, resided in the substrate‐binding pocket; and the other site was located in a region conserved in the β‐galactosidase family. These results strongly suggest that recovery of lacZ mutations at each site largely depend on the adjacent sequence context and the extent to which the mutation damages the enzymatic activity of the gene product. Environ. Mol. Mutagen. 36:301–311, 2000.

Mutation induction with UVB in mouse skin epidermis is suppressed in acute high-dose exposure.

The time and dose dependence of ultraviolet B (UVB)-induced mutant frequency (MF) in skin epidermis and dermis was studied with transgenic Muta mice harboring lambdagt10lacZ shuttle vector. Mutants of the lacZ transgene appearing in these tissues after 0.5kJ/m(2) UVB irradiation were fully expressed in 3-7 days, and the frequencies of those fully expressed mutants were maintained for at least the following 3 weeks. These fully expressed MFs increased dose-dependently, with the initial slope for the epidermis four times larger than that for dermis. Surprisingly, in epidermis, an inhibition of the dose-dependent mutation induction was evident after irradiation above 0.5kJ/m(2) UVB, lowering the increment more than eight-fold, while such suppression was not observed in dermis. This anticarcinogenic epidermal response disappeared with dose fractionation when the fractions were delivered at 4-week intervals, but not when delivered every day, showing that the induced mutation suppression is maintained under continual repetitive exposure, without which it expires within 4 weeks. These results suggest that repetition of heavy sun exposure at long intervals, e.g. recreational sunbathing every summer, is more likely to cause skin cancer than every day continual exposure even if the total UV doses are the same.

Molecular nature of mutations induced by a high dose of X-rays in spleen, liver, and brain of the lacZ-transgenic mouse

DNA sequences of 103 spontaneous mutants and 102 X‐ray‐induced mutants of the lacZ transgene from spleen, liver, and brain of the Muta™Mouse were examined and compared to elucidate characteristics of radiation‐induced mutations in vivo. The radiation‐induced mutants were isolated from genomic DNA of each tissue collected at 3.5 days after 200 Gy of whole body irradiation. Base substitution was predominant (80% or more) in nonirradiated tissues, while deletion was prevalent (about 55%) in irradiated tissues. The other types of mutation appeared at similar frequencies in both control and irradiated tissues. The size of the deletions was smaller than 438 nucleotides, with a predominance of one basepair deletions in both control and irradiated tissues. A close look at the nucleotides at the deletion endpoints revealed that many of the radiation‐induced deletions did not have repeated sequences at the break point termini, whereas all deletions found in unirradiated tissues showed one or more bases of repeated sequences at the termini. Further, eight complex‐type deletion mutations were found only in irradiated tissues. Comparison among the three types irradiated tissue did not reveal any tissue‐specificity. The data indicate that the molecular nature of mutations induced in tissues with ionizing radiation is different from that of spontaneous mutations. Environ. Mol. Mutagen. 34:97–105, 1999