DNA replication in eukaryotic cells is tightly controlled by a licensing mechanism, ensuring that each origin fires once and only once per cell cycle. We demonstrate that the ataxia telangiectasia and Rad3 related (ATR)–mediated S phase checkpoint acts as a surveillance mechanism to prevent rereplication. Thus, disruption of licensing control will not induce significant rereplication in mammalian cells when the ATR checkpoint is intact. We also demonstrate that single-stranded DNA (ssDNA) is the initial signal that activates the checkpoint when licensing control is compromised in mammalian cells. We demonstrate that uncontrolled DNA unwinding by minichromosome maintenance proteins upon Cdt1 overexpression is an important mechanism that leads to ssDNA accumulation and checkpoint activation. Furthermore, we show that replication protein A 2 and retinoblastoma protein are both downstream targets for ATR that are important for the inhibition of DNA rereplication. We reveal the molecular mechanisms by which the ATR-mediated S phase checkpoint pathway prevents DNA rereplication and thus significantly improve our understanding of how rereplication is prevented in mammalian cells.

A reduced growth hormone (GH)-insulin-like growth factor (IGF)-1 axis is associated with an extension of lifespan in laboratory rodents. Several phenotypes of such animal models resemble those induced by caloric restriction (CR). Using a transgenic male Wistar rat model whose GH-IGF-1 axis was moderately suppressed by overexpression of the antisense GH transgene (tg), we elucidated a relationship between the effects of a reduced GH-IGF-1 axis and CR for some biomarkers of aging, lifespan, and pathologies. Heterozygous (tg/-) rats fed ad libitum (AL) had a dwarf phenotype similar to that of control nontransgenic (-/-) rats subjected to 30% CR from 6 wk of age. Both the reduced GH-IGF-1 axis and CR extended lifespan to a similar extent, although the effect of CR seemed to be greater. There was an additive effect of CR to lifespan extension when tg/- rats were subjected to CR. Pathologic analyses indicated that the preventive effect of CR on selected diseases was greater than that of the reduced GH-IGF-1 axis. The present study suggests that CR affects aging and longevity by mechanisms other than suppression of the GH-IGF-1 axis, although CR might exhibit its effects partly through the reduced GH-IGF-1 axis.

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Forkhead box O (Foxo) transcription factors may be involved in the salutary effect of dietary restriction (DR). This study examined the role of Foxo3 in lifespan extension and cancer suppression in DR mice. Wild-type (WT) and Foxo3-knockout heterozygous (+/–) and homozygous (–/–) mice were subjected to a 30% DR regimen initiated at 12 weeks of age. Control mice were fed ad libitum (AL) throughout the study. In contrast to WT mice, DR did not significantly extend the lifespan of Foxo3+/– or Foxo3–/– mice. However, DR reduced the prevalence of tumors at death in WT, Foxo3+/–, and Foxo3–/– mice. These results indicate the necessity of Foxo3 for lifespan extension but not cancer suppression by DR. The findings in Foxo3+/– mice contrast with those in Foxo1+/– mice reported previously by our laboratory suggest differential regulation of cancer and lifespan by DR via Foxo1 and Foxo3.

The longer life span in dwarf mice suggests that a reduction in the growth hormone (GH)-insulin-like growth factor (IGF)-1 axis retards aging and extends the life span in mammals. We tested this hypothesis in a transgenic strain of rats whose GH gene was suppressed by an anti-sense GH transgene. Male rats homozygous for the transgene (tg/tg) had a reduced number of pituitary GH cells, a lower plasma concentration of IGF-1, and a dwarf phenotype. Heterozygous rats (tg/-) had an intermediate phenotype in plasma IGF-1, food intake, and body weight between tg/tg and control (-/-) rats. The life span of tg/tg rats was 5 to 10% shorter than -/- rats. In contrast, the life span of tg/- rats was 7 to 10% longer than -/- rats. Pathological analysis suggested that neoplasms caused earlier death in tg/tg rats; in contrast, tg/- rats had reduced nonneoplastic diseases and a prolonged life span. Immunological analysis revealed a smaller population and lower activity of splenic natural killer cells in tg/tg rats. The results of the present study support the hypothesis, but suggest that there is an optimal level of the GH-IGF-1 axis to maximize survival in mammals.

The whole genome scan for quantitative trait loci (QTLs) specifying peak bone mass was performed with the F2 intercrosses of SAMP6, an established murine model of senile osteoporosis, exhibiting a significantly lower peak bone mass, and SAMP2, exhibiting a higher peak bone mass. Cortical thickness index (CTI), a parameter of bone mass of femurs, was measured in 488 F2 progeny at 4 months of age, when the animals attained peak bone mass by microphotodensitometry. Genetic markers were typed at 90 loci spanning all chromosomes except the Y. By interval mapping of 246 male F2 mice, two loci were identified with significant linkage to peak bone mass, one on Chromosome (Chr) 11 and another on Chr 13, with a maximum lod score of 10.8 (22.2% of the total variance) and 5.8 (10.0%), respectively. Another locus on the X Chr was suggestive of a QTL associated oppositely with a low peak bone mass to the SAMP2 allele. This association was consistent with the distribution of peak bone mass in the F1 and F2. These findings should be useful to elucidate the genetics of osteoporosis.

Nijmegen breakage syndrome (NBS) is characterized by radiation hypersensitivity, chromosomal instability, and predisposition to cancer. Nbs1, the NBS protein, forms a tight complex with Mre11 and Rad50, and these interactions contribute to proper double-strand break repair.

SummaryThe FoxO transcription factors may be involved in the antiaging effect of calorie restriction (CR) in mammals. To test the hypothesis, we used FoxO1 knockout heterozygotic (HT) mice, in which the FoxO1 mRNA level was reduced by 50%, or less, of that in wild-type (WT) mouse tissues. The WT and HT mice were fed ad libitum (AL) or 30% CR diets from 12 weeks of age. Aging-and CR-related changes in body weight, food intake, blood glucose, and insulin concentrations were similar between the WT and HT mice in the lifespan study. The response to oxidative stress, induced by intraperitoneal injection of 3-nitropropionic acid (3-NPA), was evaluated in the liver and hippocampus at 6 months of age. Several of the selected FoxO1-target genes for cell cycle arrest, DNA repair, apoptosis, and stress resistance were up-regulated in the WT-CR tissues after 3-NPA injection, while the effect was mostly diminished in the HT-CR tissues. Of these gene products, we focused on the nuclear p21 protein level in the liver and confirmed its up-regulation only in the WT-CR mice in response to oxidative stress. The lifespan did not differ significantly between the WT and HT mice in AL or CR conditions. However, the antineoplastic effect of CR, as indicated by reduced incidence of tumors at death in the WT-CR mice, was mostly abrogated in the HT-CR mice. The present results suggest a role for FoxO1 in the antineoplastic effect of CR through the induction of genes responsible for protection against oxidative and genotoxic stress.