Mutation Research/DNAging最新文献

DNA polymerase α/primase (pol α) isolated from fibroblasts established from a 66-year-old human donor (GM3529) exhibited decreased specific activity compared with pol α from either fetal-derived fibroblasts (W138), or pSV3.neo-transformed GM3529 fibroblasts. The pol α specific activity decrease was correlated with a decreased proliferative capacity frequently seen in cells from aged donors. Pol α isolated from pSV3.neo-transformed GM3529 cells (GM3529T) exhibited a single isoform with about 10-fold higher specific activity than pol α from GM3529 cells. GM3529T pol α was immunoreactive with both anti-pol α and anti-SV40 large tumor antigen. Polymerases from GM3529 and GM3529T cells were treated with a pol α accessory protein, αAP, isolated from L1210 cells. Pol α from GM3529T cells showed no increase in activity in the presence of αAP, while pol α isolated from GM3529 cells exhibited about an 8-fold increase in activity after treatment with αAP. Double stranded SV40 DNA containing multiple ori sequences exhibited a greater decrease in electrophoretic mobility in the presence of GM3529T pol α. In the presence of pol α from either GM35229 or GM3529T cells SV40 dsDNA exhibited a decrease in electrophoretic mobility, and in each instance addition of αAP resulted in an even greater decrease in DNAm mobility. These data indicate that αAP increased pol α binding to SV40 dsDNA, or that αAP bound the DNA in addition to previously bound pol α. GM3529 pol α also bound non-specific, non-SV40, dsDNA, whereas GM3529T pol α with associated TAg did not bind the non-viral dsDNA unless αAP was added to the preparation. While not all human diploid fibroblast cell lines derived from aged human donors necessarily exhibit decreased proliferative capacity compared with cells from young donors, decreased specific activity associated with a decline in cellular DNA synthesis is typical of pol α from cells derived from aged human donors. We suggest that a decrease in endogenous αAP interaction with pol α may account, in part, for the loss of DNA binding affinity and specific activity of pol α from GM3529 cells derived from an aged donor.

All strains of the filamentous fungus Podospora anserina are characterized by a well defined life span. Senescence is controlled by nuclear and extranuclear genetic traits. In order to test whether or not the ends of the chromosomes of this ascomycete shorten during senescence and thus tolemere shortening may be linked to the well analyzed, age-related reorganizations of the mitochondrial DNA (mtDNA), we analyzed the genomic DNA of P. anserina wild-type strain s. We found that, although the mtDNA becomes reorganized when cultures age, the tolemeres remain constant suggesting that tolemere shortening does not play a major role in normal aging of this particular biological system.

Evolution theory indicates that ageing is caused by progressive accumulation of defects, since the evolutionary optimal level of maintenance is always below the minimum required for indefinite survival. Evolutionary theories also suggest that multiple processes are operating in parallel, but unfortunately they make no predictions about specific mechanisms. To understand and evaluate the many different mechanistic theories of ageing which have been proposed, it is therefore important to understand and study the network of maintenance processes which control cellular homeostatis.

In this paper we describe a Network Theory of Ageing which intergrates the contributions of defective mitochodria, aberrant proteins, and free radicals to the ageing process, and which includes the protective effects of antioxidant enzymes and proteolytic scavengers. The model simulations not only cofirm and explain many experimental, age related findings like an increase in the fraction of inactive proteins, a significant rise in protein half-life, an increase in the amount of damaged mitochondria, and a drop in the energy generation per mitochondrion, but they also show interactions between the different theories which could not have been observed without the network approach. In some simulations, for example, the mechanism of the final breakdown seems to be a consequence of the cooperation of mitochondrial and cytoplasmic reactions, the mitochondria being responsible for a long term, gradual change which eventually triggers a short lived cytoplasmic error loop.

Spermatid micronuclei (MN) from Armenian hamsters in differebnt age groups were compared with regard to frequencies and kinetochore status (presence or absence) as determined with immunofluorescent staining. Six thousand cells analyzed from each of fifteen young animals (3 months) revealed a group mean frequency of 0.45 MN/1000 spermatids; kinetochor staining was uniformly negative. Six thousand cells scored from each of fifteen older animals (2 years)_revealed a group mean frequency of 1.00 MN/1000 spermatids. Most of the MN in these animals were negative for kinetochor staining, although a signiificant representation of MN with positive kinetochore staining was also observed. The results indicate that frequencies of spermatid MN increase with advancing age, and suggest that the increase is due to significant elevations in both chromosome breakage and chromosome loss.

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals beloning to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS · MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase ni mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ae, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

Fluorometric analysi of DNA unwinding (FADU) is a fast and reliable method for detecting single strand DNA breaks as an index of DNA damage induced by clastogenic agents. A study of damage detected by FADU was conducted on DNA extracted from peripheral blood lymphocytes of 128 healthy nonsmoking regular donors (ranging in age from 19 to 67 years) and from 5 umbilical cord blood samples. DNA damage was measured as percentage of unwound DNA after alkalinization. Statistical analyses, both parametric (Pearson r correlation coefficient, b regression coefficient, ANOVA) and nonparametric (Kruskal-Wallis H test, Spearman r_s rank correlation coefficient) support a significant correlation between age of donors and amount of DNA damage. The same results are found when adult donors are divided in four age classes and ANOVA test performed among the mean percentages of unwound DNA of each class. Furthermore, donors of the same age belonging to different blood groups (A, B. AB and O) do not show any difference in DNA damage detected by FADU.

We have determined the frequency and spectrum of spontaneous mutations at the hprt locus in LovO, HCT116, LS180 and DLD-1 colon carcinoma cell lines exhibiting microsatellite genetic instability. Each cell line has a different mutator gene. LoVo and HCT116 cells have mutated hMSH2 and hMLH1 genes, respectively, which account for the majority of hereditary non-polyposis colorectal cancer (HNPCC). LS180 cells are wild type for these genes and also for hPMS1 and hPMS2 mismatch repair genes. DLD-1 cells harbor a mutated GTBP mismatch binding factor and a mutated DNA Polymerase δ. The mutation rate at the hprt locus was several hundred fold higher in these cell lines relative to control cell lines without microsatellite instability. The mutations were frameshifts (deletions and insertions of a single nucleotide in short repeats) and single base substitutions (transversions and transitions). Some mutations were shared by these fouor cell lines. However, every cell line also exhibited a distinctive spectrum of mutations suggesting that each mutator gene induces a particular mutator phenotype. These results also suggest that the frequency and spectrum of somatic mutations in tumor cells of the microsatellite mutator phenotype may have diagnostic applications to discriminate among the diverse underlying mutator genes.

The expression of DNA polymerase α (pol α) was studied in human fibroblast lines WI38 (fetal lung) and GM3529 (skin, established from a 66 yr old donor), and their Simian virus 40 (SV40) large tumor antigen (TAg)-transformed corollaries, 2RA and 2-1 respectively. Both SV40-transformed and pSV3.neo (SV40-derived plasmid)-transformed cells express TAg, a virally encoded protein not expressed by the normal parent cell lines. Northern blot hybridization studies showed increased recovery of pol α mRNA from transformed cells compared with normal cells. This increase was correlated with increased pol α mRNA transcription as determined by nuclear run-on assays. Northern blot analyses also showed an increase in the instability of translationally active pol α mRNA in transformed cells. The results suggest that TAg, in addition to its dsDNA binding, pol α binding, retinoblastoma protein binding and helicase activities, may be involved either directly or indirectly in regulation of the steady state mRNA levels of pol α at the transcriptional level in both fetal and aged donor-derived transformed fibroblasts.

Damage to DNA seems to be involved in aging and the etiology of age-associated degenerative disease. The purpose of this study is to examine changes in DNA damage during aging. Am oxidized nucleoside, 8-hydroxy-2′-deoxyguanosine (8-OHdG), is a proposed biomarker for DNA damaged by oxidative stress. The content of 8-OHdG in nuclear DNA isolated from brain, heart, liver, and kidneys of male Fischer 344 rats of different ages was measured. 8-OHdG can be detected selectivity and sensitivity at the fmol level by high performance liquid chromatography-electrochemical detection at an applied ptential of +350 mV. The amount of 8-OHdG, expressed as the ratio oto deoxyguanosine in nuclear DNA, in heart, liver, and kidney remained steady from 2 to 24 months and then increased progressively. The content of 8-OHdG in teh DNA in brain showed no changes from 2 to 27 months, but was significantly higher in 30 month-old rats. There was a significant 2-fold increase in the amount of 8-OHdG in the nuclear DNA of all organs tested in 30 month-old rats as compared to 2–24 month-old rats. These results indicate that the accumulation of 8-OHdG in the DNA of rat organs begins at ages above 24 months.