Tetsuya Ono

Age-associated changes in DNA methylation and mRNA level of the c-myc gene in spleen and liver of mice

Age-associated changes in DNA structure and mRNA level were studied for the c-myc gene in spleen and liver of mice at 2, 14 and 26 months of age. Neither amplification nor rearrangement of the gene was detected in either tissue at any age. However, a significant alteration was observed in the methylation profile. The profile of the gene and its vicinity was determined using various methylation-sensitive restriction enzymes. In both tissues, the gene had an unmethylated domain ranging from -2 kb upstream of the 5 end of the first exon to the 3 end of the first intron. It was flanked by partially methylated regions, where age-dependent changes as well as tissue specificity were observed. In the spleen, age-related hypomethylation was observed at both the 3 and 5 sides of the domain. In contrast, hypermethylation was found in the liver only at the 3 side. The steady-state level of c-myc mRNA showed a drastic decrease in liver from youth to middle age, while splenic mRNA changed little. The correlation between the changes of mRNA and DNA methylation is discussed.

Prostacyclin Production in Cultured Endothelial Cells is Highly Sensitive to Low Doses of Ionizing Radiation

In an attempt to detect if low doses of ionizing radiation affect the physiological function of blood vessels, the effect of prostacyclin production was examined using cultured endothelial cells. Irradiation with 5 cGy suppressed the production to 53% of the levels in unirradiated control cells. The PGI2 production decreased with an increase in the dose, showing a maximal decrease at 100 cGy without further decrease at 200 cGy. The suppressive effect lasted for 15 min after irradiation and disappeared later. Preincubation with alpha-tocopherol abrogated the suppressive effect completely, although preincubation with ascorbic acid showed little effect. These results suggest that low dose irradiation inhibits the prostacyclin production by changing the cellular membranes transiently.

Endogenous virus genomes become hypomethylated tissue—Specifically during aging process of C57BL mice

In an attempt to find out a cause for age-dependent derepression of endogenous viruses, extents of DNA methylation at the endogenous B- and C-type ecotropic viruses in brain, liver and spleen of C57BL/6NJc1 were examined at three ages, newborn, young adult and old. Both endogenous viruses showed a slight but significant tissue-specific either hypo- or hypermethylation during post-natal developmental phase in the three tissues. After maturation, however, no such change was detectable at most of the sites examined. The exceptions were C-type ecotropic virus in brain and B-type virus in spleen, where the age-dependent decreases of methylation were observed. The changes seemed to be continuations of preceding developmental hypomethylation. They indicated that the hypomethylation could be one of the causes for the age-dependent derepression of endogenous virus. It was further suggested that a mechanism to stop the developmental changes of DNA methylation at the maturation of individuals would be important in considering the reasons for the changes in senescent phase.

Methylation of the c―myc gene changes during aging process of mice

The degree of methylation at c-myc proto-oncogene was found to change during aging process of mice by the use of methylation-sensitive restriction enzymes. The spleen DNA showed hypomethylation as mice aged, while hypermethylation was observed in the liver DNA. The brain DNA on the contrary revealed no appreciable difference between young and old mice. When the DNAs were examined at actin and dihydrofolate reductase (DHFR), no significant change was observed. It suggests that an age-related change of oncogene structure may be one of the factors which are related to an age-associated increase of cancer incidence rate.

Absence of gross change in primary DNA sequence during aging process of mice

The possibility of age-associated changes in DNA sequence in terms of amplification and rearrangement was examined in mouse spleen, liver and brain using the method of Southern transfer and filter hybridization. The DNA regions studied were at and around nine cloned sequences, most of which are known to move or amplify in certain situations. No detectable age-associated change, however, was observed in all DNA regions studied. These results suggest that widespread DNA sequence rearrangements or amplifications do not occur during the ageing process in mice.

Effects of energy restriction on age-associated changes of DNA methylation in mouse liver.

DNA methylation is known to change with age in several mammalian species. Here we have examined the effect of dietary energy restriction on this age-associated change in liver DNA of C3H/SHN mice. The total 5-methyldeoxycytidine level in the genome decreased slightly soon after energy restriction started. The effect, however, diminished with time and no appreciable difference was detected at middle and old ages. The degree of methylation at the c-myc gene, on the other hand, was not affected by energy restriction at early periods, but the age-dependent alterations at later ages were repressed. This is a new finding to show that DNA methylation is one of the molecular indices of aging affected by energy restriction. It suggests an importance of DNA methylation in the aging process.

Comparison of age-associated changes of c-myc gene methylation in liver between man and mouse

Age-associated changes in DNA methylation of the c-myc gene in liver were compared between humans and mice which have large differences in aging rate. Although the overall methylation profiles of the gene and their age-related changes were found to be similar, the rate of change was much slower in humans than in mice. It suggests that the age-associated alteration of c-myc gene methylation is closely related to biological aging rather than to chronological aging.

Effect of Recombinant Human Granulocyte Colony-Stimulating Factor on Survival in Lethally Irradiated Mice

Repeated injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF) to lethally irradiated mice increased the rate of animal survival. Dose modification factor was 1.20 when 4.5 micrograms/mouse of rhG-CSF was given daily for 14 days after whole body irradiation. Haematological examinations revealed that rhG-CSF increased the number of blood-circulating leukocytes, neutrophils, monocytes and erythrocytes, but not that of lymphocytes and thrombocytes. Spleen weight and number of endogenous spleen colonies were also increased by rhG-CSF treatment compared with the values for mice irradiated only.

Comparison of the Age- and Tumor-Associated Changes in the c-myc Gene Methylation in Mouse Liver

Study of DNA methylation of the c-myc gene in liver tumors of BCF1 mice treated with various agents revealed frequent alteration from that in normal liver. The alteration, however, was complex and showed either an increase or a decrease of methylation to various degrees. On the other hand, the tumors obtained from middle-aged C3H/He mice, which develop liver tumors spontaneously at a high incidence rate starting in middle age, showed predominantly increases of methylation in the c-myc gene, while the large tumors found in older mice revealed decreases. The normal part of the liver showed a slight gradual age-dependent increase. These suggest that hypermethylation of the c-myc gene is common to aging and early tumor development in liver. Thus the alteration of DNA methylation seems to be a good clue to investigate why the tumor incidence rate increases rapidly as individuals grow older.

Prevention of Radiation-induced Bacteraemia by Post-treatment with OK-432 and Aztreonam

The effects of combined treatment with OK-432, an immunomodulator prepared from Streptococcus haemolyticus, and aztreonam, a monobactum antibiotic, in the prevention of radiation-induced bacteraemia and mortality were examined in ICR-MCH mice irradiated with 9.5 Gy. The organisms recovered from the irradiated mice were Streptococcus faecalis and Proteus mirabilis. Treatment with aztreonam reduced the incidence of mice infected with Proteus mirabilis (p < 0.01), but it showed no efficacy on Streptococcus faecalis. OK-432 could reduce the frequency of bacteraemia attributed to both organisms (p < 0.05). Combined treatment with OK-432 and aztreonam further decreased the incidence of bacteraemia by both organisms; no organisms were recovered at 14 days following irradiation. The survival rate at 30 days following irradiation was 80% in mice treated with OK-432 plus aztreonam and 55% with OK-432 alone, while it was 0% in the groups treated with aztreonam or saline alone. These results indicated that combined treatment with OK-432 and a suitable antibiotic such as aztreonam is more effective than OK-432 or aztreonam alone.