Mutation Research/DNAging最新文献

There are many methods of inference in common use in biology that are based on population sampling, including such diverse areas as sampling organisms to determine the population structure of an ecosystem, sampling a set of DNA sequences to infer evolutionary history, sampling genetic loci to build a genetic map, sampling differentially expressed genes to find phenotypic markers, and many others. Recently developed PCR-based methods for nucleic acid fingerprinting can be used as sampling tools with general applicability in molecular biology, evolution and genetics. These methods include arbitrarily primed PCR (AP-PCR; Welsh and McClelland, 1990) and random amplified polymorphic DNA (RAPD; Williams et al., 1990) for the fingerprinting of DNA, and RNA arbitrarily primed PCR (RAP-PCR; Welsh et al., 1992a) and differential display (DD; Liang and Pardee, 1992) for the fingerprinting of RNA. Novel ways of looking at genetic control are facilitated by the high data-acquisition capabilities of the fingerprinting methods. In this article, we review some of the applications of DNA fingerprinting to the study of mutagenesis, and of RNA fingerprinting to the study of normal and abnormal signal transduction. We propose that these fingerprinting approaches may also have applications in the study of senescence and aging.

Sister chromatid exchanges (SCEs) and cell cycle kinetics were estimated in mitogen stimulated human lymphocytes from a selected group of healthy individuals. Data were examined to evaluate the relationship between SCE frequencies and cell cycle kinetics with donor's age, sex and smoking habit. No regular relationship was observed between the means SCE frequencies and donor's age, although significant differences were observed between the age groups. Correlation of dispersion coefficient (H) of SCE with donor's age were significant in male and female populations. For cell cycle kinetics, a highly significant age-dependent depression in replicative index (RI) was observed. Female donors possessed a slightly higher SCE frequency and RI, although the variations between the two sexes were not significant. Smoking habit resulted in a significant enhancement of SCEs.

DNA modification by cytosine methylation has received considerable interest in the context of mammalian cell differentiation but is discussed controversially with respect to cellular aging. As the expression of c-myc affects strongly cellular aging and terminal differentiation, we have analysed the sequence-specific methylation pattern of the c-myc gene during proliferative aging in vitro of human embryonic fibroblasts. In this study, both, 5-methylcytidine sensitive restriction enzymes as well as genomic sequencing were used. The overall methylation pattern was found essentially stable during proliferative aging. However, specific hypermethylation of exon II during aging was observed. Futhermore, one specific cytidine located in the consensus sequence of the DNA binding factor PEBP2 was found completely methylated during most of the course of proliferative aging of the cells but became demethylated as the cells reached the end of their proliferative life span. Our results indicate the importance of establishing the sequence-specific changes of the methylation pattern of the genome during in vitro aging.

LIO rats were exposed to s.c. injections (3.2. mg) of a synthetic analogue of thymidine, 5-bromo-2′-deoxyuridine (BrfUrd) on the 1st, 3rd, 7th and 21st days of life and at the age of 3 or 15 months they were i.v. injected with N-nitrosomethylurea (NMU) at a single dose of 10 or 50 mg/kg or with solvent. It was shown that early neonatal exposure to BrdUrd was followed by the increase in the incidence of tumor development and by the decrease of their latency. The carcinogenic effect of NMU alone correlated with the dose of the carcinogen in 3-month-old rats and did not correlate with dose in the 15-month-old ones. As compared to the 3-moth-old rats, the incidence of total and malignant tumors and tumors of some localization was decreased in the elder ones, but survival of tumor-bearing rats was decreased in the elder group as compared to the younger one. These data suggests the age-related decrease in both the carcinogenic effect of NMU and in the number of events whic are necessary for a tumor development. The exposure to BrdUrd was followed by the increase in the susceptibility of rats to subsequent carcinogenic effect of NMU injected at the doses of 10 or 50 mg/kg into 3- and 15-month-old rats, mostly to the tissues being target to NMU. Our data have demonstrated that the exposure to BrdUrd in the eraly life was followed by the irreversible initiating effect which persists over a long time in a several tissues.

In this paper, we present various arguments supporting the hypothesis that reactive oxygen species (ROS) could be responsible for the modulation of various cellular functions, besides their well known toxic effects.

We first review the recent evidence indicating that ROS are able to modulate genome expression through specific and precise mechanisms during cell activation. The role of the nitrogen reactive radicals such as nitric oxide is separately analyzed because of its specific role in the nervous and vascular systems. The action of the other ROS on gene activation will then be reviewed by first looking at their possible involvement in the activation of transcription factors like NF-κB. Arguments will then be developed in favor of the implication of the ROS in the cellular effects of PMA, TNF-α and other cytokines on the modulation of the genetic expression. Possible mechanisms will be presented for linking the production of the ROS with cell activation. In a general way we postulate that ROS can play a role of secondary messengers in several cell responses to external stimuli.

In the second part of the paper, we will examine the long term influence of ROS and their possible roles in cellular aging. Different links exist between ROS and aging and the relationship between them is probably indirect. We propose to consider the effect of ROS as one of the multiple challenges that cells have to face, the cell being considered as a global system which must optimize its energy expenditure for carrying out its basic functions such as turnover, differentiated phenotype functions, multiplication, defense and repair processes. This thermodynamic point of view will help to understand the effect of low ROS stresses, among others, on accelerated aging.

Three-month-old male and 3-month-old female LIO rats as well as 25-month-old males and 3-month-old females were mated and at the age of 3 months their progeny were exposed to a single intravenous injection of N-nitrosomethylurea (MNU) at the dose of 20 mg/kg of body weight or solvent. Animals were under observation during 18 months after injection of the carcinogen. There was no significant difference in spontaneous tumor incidence between progeny of young and old male rats. At the same time, the susceptibility to the carcinogenic effect of NMU in the male and female progeny of old males was slightly but significantly increased in comparison to the progeny of young males. Mesenchymal kidney tumors were discovered in the NMU_treated male progeny of old males but not in the male progeny of young male rats. In females, the incidence of mesenchymal kidney tumors in the NMU-treated progeny of young and old males was 7% and 20%, respectively, and the mean survival times of these tumor-bearing rats was 4 months shorter in the last group. The data obtained are in agreement with the observation on germ-line transgeneration transmission of predisposition to carcinogenesis.

Oxidation of DNA and its components can cause genetic mutations and chromosomal instability. These changes have generally been implicated in aging. Oxidation of the methyl group of thymidine residues in DNA is known to result in the formation 5-hydroxymethyl-2′-deoxyuridine (5HmdUrd). We have utilized Bacillus subtilis phage SPO1 DNA as a model of oxidatively damaged DNA. In this phage, all thymine (Thy) residues are replaced by 5-hydroxymethyluracil (5HmUra), but the species is naturally devoid of other oxidatively-induced DNA lesions. Particular attention was paid to the behavior of 5HmUra-containing DNA as a target for several enzymes employing DNA as substrate; restriction endonucleases, dam DNA methylase and T4 DNA ligase. We noticed that susceptibility of SPO1 DNA varied when different restriction endonucleases having 5HmUra in the restriction sites were tested. Endonucleolytic cleavage brought about Sau3A proceeded as effectively with SPO1 DNA as with conventional DNA (lambda phage). The same was true when the ligation of Sau3A sites was performed with T4 DNA ligase. In contrast, both endonucleolytic cleavage and ligation were slower in SPO1 DNA, compared with lambda phage, when Taq I and T4 DNA ligase were used for restriction and ligation, respectively. We also noticed that SPO1 phage does not naturally contain N⁶-methyladenine (N⁶MeAde) opposite 5HmUra, i.e., no hydrolysis of SPO1 DNA was observed when assessed with methylation-dependent restriction endonuclease DpnI. Our results show that the presence of 5HmUra in the respective site of DNA does not, per se, prevent the activity of restriction endonucleases, ligases or DNA methylases. These data support the view that oxidation of Thy to 5HmUra in target DNA does not necessarily result in substantial deterioration in the functions of DNA processing enzymes.

Two fibroblast cell lines (PG3KT and PG1NA) deruved from Hutchinson-Gilford syndrome (progeria) cases were characterized, at various population doubling levels (PDL), with respect to the capacity of ultraviolet light (UV, mainly 254 nm wavelength)-induced unschedules DNA synthesis (UDS) and plasminogen activator-like protease activity (PA). The UDS levels in PG3KT and PG1NA cells at PDL 2–3 were only slightly less than those in normal fibroblasts. With increasing PDL. both prgeria cell lines exhibited reduction of the UDS levels and undetectable ones at PDL 9–11. Prompt and transient induction of PA was also detectable at less than PDL 5, whereas it was undetectable at higher PDL. However, the levels of UDS and PA induction were increased about 3–7 times after pretreatment with 100 IU/ml human interferon (HuIFN)-β preparations for more than 24 h prior to UV irradiation, although UDS and PA were undetectable at more than PDL 10. These results suggest that cytokines such as HuIFN-β transiently compensate for the decreases in UDS and PA inducibility in progeria cells with aging.

An accumulation of mutations on their own or together with other age-related changes may contribute to ageing and the development of age-related pathologies. The aim of this investigation wass to assess the extent of DNA mutations as a function of age in humans. The mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl-transferase (hgprt) locus was assessed in lymphocytes isolated from male volunteers in each of three age groups (35–39, 50–54 and 65–69 years). Results show that the mean MF in the 65–69 years group was approximately twice that in the 35–39 and 50–54 years (4.1/10⁶ cells, 1.9/10⁶ cells and 1.79/10⁶ cells, respectively) increasingly by about 1.33% per year, after 54 years. In addition, there was an increased frequency of chromosomal aberrations in the 65–69 years group compared to the other two age groups. The results of this investigation show an increase in DNA mutations in cultured human lymphocytes with age. Factors which may influence the extent of DNA damage in human lymphocytes are discussed.

Recent studies on human tissues have shown that the guantity of partially deleted mitochondrial DNA (mtDNA) increases with age. In this study, mtDNAs from the livers of young adult and old Wistar rats were analyzed by PCR. Evidence for partially deleted mtDNAs was found, with a 4834-bp deletion present in all animals and most easily detected in samples from senescent rats. The deletion breakpoint occurs at a 16-bp direct repeat present in the cytochrome oxidase I and ATPase 6 genes. This deletion in rats is similar in size and location to the 5.0-kb deletion observed in human mtDNA. The proportion of rat mtDNA with this 4.8-kb deletion was quantitated by a competitive PCR assay. The ratio of partially deleted mtDNA/ total mtDNA in liver mtDNA from individual 6 month old rats ranged from 5 × 10⁻⁶ to 3 × 10⁻⁵, while the ratio in 24 month old rats ranged from 8 × 10⁻⁴ 5 × 10⁻³, with a mean 100-fold increase with age. These increases are in the range observed for human mtDNA during aging. Thus senescent rats can be used as a model to study this type of mitochondrial DNA damage in aging. The method and reagents described should prove useful of the mechanism(s) underlying deletions, their significance to the aging process, and testing of various compounds or interventions for their ability to slow the process.