Caenorhabditis elegans ATR checkpoint kinase ATL-1 influences life span through mitochondrial maintenance.

ATR is highly conserved in all eukaryotes and functions as a cell-cycle nuclear checkpoint kinase. In mammals, ATR is essential whose complete absence results in early embryonic lethality and its hypomorphic mutation causes a complex disease known as Seckel syndrome. However, molecular mechanisms that cause a wide variety of symptoms including accelerated aging have remained unclear. Similarly, in the nematode Caenorhabditis elegans, a deletion mutant of ATR ortholog atl-1 appears to develop into normal adults, but their eggs do not hatch and die at early embryogenesis. Here we show that the parental worms of atl-1 defective mutant achieved longevity. Transcription levels of certain superoxide dismutase genes, sod-3 and -5 and enzymatic activity of superoxide dismutases significantly increased in the mutant. Furthermore, lipid peroxidation such as a formation of malondialdehyde was attenuated. Expressions of other genes regulated by DAF-16/FOXO transcription factor were also altered. In contrast, the mutant became hypersensitive to rotenone and ethidium bromide. Compared with the wild type the mitochondrial DNA copy number in the mutant was lesser and its proliferation is more severely inhibited in the presence of rotenone. These results suggest that C. elegans ATL-1 is involved not only in the nuclear checkpoint control but also in the mitochondrial maintenance, and its dysfunction activates mild oxidative stress response, resulting in an alteration of life span.

Innate immune genes including a mucin-like gene, mul-1, induced by ionizing radiation in Caenorhabditis elegans.

The effect of radiation on the intestine has been studied for more than one hundred years. It remains unclear, however, whether this organ uses specific defensive mechanisms against ionizing radiation. The infection with Pseudomonas aeruginosa (PA14) in Caenorhabditis elegans induces up-regulation of innate immune response genes. Here, we found that exposure to ionizing radiation also induces certain innate immune response genes such as F49F1.6 (termed mul-1), clec-4, clec-67, lys-1 and lys-2 in the intestine. Moreover, pre-treatment with ionizing radiation before seeding on PA14 lawn plate significantly increased survival rate in the nematode. We also studied transcription pathway of the mul-1 in response to ionizing radiation. Induction of mul-1 gene was highly dependent on the ELT-2 transcription factor and p38 MAPK. Moreover, the insulin/IGF-1 signal pathway works to enhance induction of this gene. The mul-1 gene showed a different induction pattern from the DNA damage response gene, ced-13, which implies that the expression of this gene might be triggered as an indirect effect of radiation. Silencing of the mul-1 gene led to growth retardation after treatment with ionizing radiation. We describe the cross-tolerance between the response to radiation exposure and the innate immune system.

Radiation biology of Caenorhabditis elegans: germ cell response, aging and behavior.

The study of radiation effect in Caenorhabditis (C.) elegans has been carried out over three decades and now allow for understanding at the molecular, cellular and individual levels. This review describes the current knowledge of the biological effects of ionizing irradiation with a scope of the germ line, aging and behavior. In germ cells, ionizing radiation induces apoptosis, cell cycle arrest and DNA repair. Lots of molecules involved in these responses and functions have been identified in C. elegans, which are highly conserved throughout eukaryotes. Radiosensitivity and the effect of heavy-ion microbeam irradiation on germ cells with relationship between initiation of meiotic recombination and DNA lesions are discussed. In addition to DNA damage, ionizing radiation produces free radicals, and the free radical theory is the most popular aging theory. A first signal transduction pathway of aging has been discovered in C. elegans, and radiation-induced metabolic oxidative stress is recently noted for an inducible factor of hormetic response and genetic instability. The hormetic response in C. elegans exposed to oxidative stress is discussed with genetic pathways of aging. Moreover, C. elegans is well known as a model organism for behavior. The recent work reported the radiation effects via specific neurons on learning behavior, and radiation and hydrogen peroxide affect the locomotory rate similarly. These findings are discussed in relation to the evidence obtained with other organisms. Altogether, C. elegans may be a good "in vivo" model system in the field of radiation biology.

Localization of X-ray cross complementing gene 1 protein in the nuclear matrix is controlled by casein kinase II-dependent phosphorylation in response to oxidative damage.

Base excision repair/single strand break repair (BER/SSBR) of damaged DNA is a highly efficient process. X-ray cross complementing protein 1 (XRCC1) functions as a key scaffold protein for BER/SSBR factors. Recent work has shown that XRCC1 forms dense foci at sites of DNA damage in a manner dependent on casein kinase II (CK2) phosphorylation. To investigate the mechanism underlying foci formation, we analyzed the subnuclear localization and phosphorylation status of XRCC1 during the repair process by biochemical fractionation of HeLa cellular proteins. The localization was also verified by in situ extraction of the fixed cells. In unchallenged cells, XRCC1 was primarily found in the chromatin fraction in a highly phosphorylated form; in addition, a minor population (10-15%) existed in the nuclear matrix (NM) with no or marginal phosphorylation. After hydrogen peroxide treatment, hyperphosphorylated XRCC1 appeared in the NM and accordingly, those in the chromatin fraction decreased. Foci formation and changes in XRCC1 distribution could be abolished by the knockdown of CK2, the expression of a non-phosphorylatable version of XRCC1, or the inhibition of poly-ADP ribosylation at the damage sites. Other BER factors, like DNA polymerase beta, were also found to accumulate in the NM after hydrogen peroxide-induced DNA damage, although its association with the NM seemed relatively weak. Our results suggest that the constitutive phosphorylation of XRCC1 in the chromatin and its DNA damage-induced recruitment to the NM are critical for foci formation, and that the core reactions of BER/SSBR may occur in the NM.

C. elegans RNAi space experiment (CERISE) in Japanese Experiment Module KIBO.

We have started a space experiment using an experimental organism, the nematode Caenorhabditis elegans, in the Japanese Experiment Module, KIBO, of the International Space Station (ISS). The specimens were boarded by space shuttle Atlantis on mission STS-129 which launched from NASA Kennedy Space Center on November 16, 2009. The purpose of the experiment was several-fold: (i) to verify the efficacy of RNA interference (RNAi) in space, (ii) to monitor transcriptional and post-translational alterations in the entire genome in space, and (iii) to investigate mechanisms regulating and countermeasures for muscle alterations in response to the space environment. In particular, this will be the first study to utilize RNAi in space.

Maintenance of mitochondrial DNA by the Caenorhabditis elegans ATR checkpoint protein ATL-1.

Here we show that inactivation of the ATR-related kinase ATL-1 results in a significant reduction in mitochondrial DNA (mtDNA) copy numbers in Caenorhabditis elegans. Although ribonucleotide reductase (RNR) expression and the ATP/dATP ratio remained unaltered in atl-1 deletion mutants, inhibition of RNR by RNAi or hydroxyurea treatment caused further reductions in mtDNA copy number. These results suggest that ATL-1 functions to maintain mtDNA independently of RNR.

The effect of high strength static magnetic fields and ionizing radiation on gene expression and DNA damage in Caenorhabditis elegans.

Magnetic resonance imaging with high static magnetic fields (SMFs) has become widely used for medical imaging purposes because SMFs cause fewer genotoxic side effects than ionizing radiation (IR). However, the effect of exposure to high SMFs on global transcription is little understood. We demonstrate that genes involved in motor activity, actin binding, cell adhesion, and cuticles are transiently and specifically induced following exposure to 3 or 5 T SMF in the experimental model metazoan Caenorhabditis elegans. In addition, transient induction of hsp12 family genes was observed after SMF exposure. The small-heat shock protein gene hsp16 was also induced but to a much lesser extent, and the LacZ-stained population of hsp-16.1::lacZ transgenic worms did not significantly increase after exposure to SMFs with or without a second stressor, mild heat shock. Several genes encoding apoptotic cell-death activators and secreted surface proteins were upregulated after IR, but were not induced by SMFs. Real-time quantitative RT-PCR analyses for 12 of these genes confirmed these expression differences between worms exposed to SMFs and IR. In contrast to IR, exposure to high SMFs did not induce DNA double-strand breaks or germline cell apoptosis during meiosis. These results suggest that the response of C. elegans to high SMFs is unique and capable of adjustment during long exposure, and that this treatment may be less hazardous than other therapeutic tools.

Caenorhabditis elegans par2.1/mtssb-1 is essential for mitochondrial DNA replication and its defect causes comprehensive transcriptional alterations including a hypoxia response.

DNA polymerase gamma and mtSSB are key components of the mtDNA replication machinery. To study the biological influences of defects in mtDNA replication, we used RNAi to deplete the gene for a putative mtSSB, par2.1, in Caenorhabditis elegans. In previous systematic RNAi screens, downregulation of this gene has not caused any clearly defective phenotypes. Here, we continuously fed a dsRNA targeting par2.1 to C. elegans over generations. Seventy-nine percent of F1 progeny produced 60-72 h after feeding grew to adulthood but were completely sterile, with an arrest of germline cell proliferation. Analyses of mtDNA copy number and cell cytology indicated that the sterile hermaphrodites had fewer mitochondria. These results indicated that par2.1 essentially functions for germline cell proliferation through mtDNA replication; we therefore termed it mtssb-1. Comprehensive transcriptional alterations including hypoxia response induction dependent on and independent of hif-1 function, occurred by RNAi depletion of mtssb-1. Treatment with ethidium bromide, which impairs mtDNA replication and transcription, caused similar transcriptional alterations. In addition, the frequency of apoptosis in the germline cells was reduced in fertile progeny with a partial RNAi effect. These suggest that RNAi depletion of C. elegans mtssb-1 is useful as a model system of mitochondrial dysfunction.

Inhibition of ocular angiogenesis by diced small interfering RNAs (siRNAs) specific to vascular endothelial growth factor (VEGF).

The effects of diced small interfering RNAs (siRNAs) designed for vascular endothelial growth factor (VEGF) on the expression of VEGF in human retinal pigment epithelial cell line ARPE-19 cells in vitro and on corneal angiogenesis in vivo were examined. The exposure to diced siRNAs significantly reduced the VEGF mRNA expression in ARPE-19 cells with minimal toxicity. In suture-induced corneal angiogenesis models, diced siRNAs minimized the severity of angiogenesis. Histological analysis displayed no particular tissue damage in the conjunctiva where siRNA was injected. The approach using diced siRNAs can be a new tool for various neovascular ocular diseases.

The Arg280His polymorphism in X-ray repair cross-complementing gene 1 impairs DNA repair ability.

The contribution of three single nucleotide polymorphisms (SNPs) that substitute amino acids in the X-ray repair cross-complementing gene 1 (XRCC1) protein, Arg194Trp (R194W), Arg280His (R280H), and Arg399Gln (R399Q), to the risk of various types of cancers has been extensively investigated by epidemiological researches. To investigate whether two of these polymorphisms directly influence their repair ability, we established Chinese hamster ovary (CHO) EM9 cell lines transfected with XRCC1(WT), XRCC1(R194W), or XRCC1(R280H) genes and analyzed the DNA repair ability of these cells. The EM9 cells that lack functional XRCC1 proteins exhibit severe sensitivity to methyl methanesulfonate (MMS). Introduction of the human XRCC1(WT) and XRCC1(R194W) gene to EM9 cells restored the MMS sensitivity to the same level as the AA8 cells, a parental cell line. However, introduction of the XRCC1(R280H) gene partially restored the MMS sensitivity, resulting in a 1.7- to 1.9-fold higher sensitivity to MMS compared with XRCC1(WT) and XRCC1(R194W) cells at the LD(50) value. The alkaline comet assay determined diminished base excision repair/single strand break repair (BER/SSBR) efficiency in XRCC1(R280H) cells as observed in EM9 cells. In addition, the amount of intracellular NAD(P)H decreased in XRCC1(R280H) cells after MMS treatment. Indirect immunofluorescence staining of the XRCC1 protein showed an intense increase in the signals and clear foci of XRCC1 in the nuclei of the XRCC1(WT) cells, but a faint increase in the XRCC1(R280H) cells, after MMS exposure. These results suggest that the XRCC1(R280H) variant protein is defective in its efficient localization to a damaged site in the chromosome, thereby reducing the cellular BER/SSBR efficiency.

Efficient repair of DNA damage induced by heavy ion particles in meiotic prophase I nuclei of Caenorhabditis elegans.

The effects of heavy ion particle irradiation on meiosis and reproductive development in the nematode Caenorhabditis elegans were studied. Meiotic pachytene nuclei are significantly resistant to particle irradiation by the heavy ions carbon and argon, as well as to X-rays, but not UV, whereas diplotene to diakinesis stage oocytes and early embryonic cells are not. Chromosomal abnormalities appear in mitotic cells and in maturing oocytes irradiated with heavy ion particles during the diplotene to the early diakinesis stages, but not in oocytes irradiated during the pachytene stage. The pachytene nuclei of ced-3 mutants, which are defective in apoptosis, are similarly resistant to ionizing radiation, but pachytene nuclei depleted for Ce-atl-1 (ataxia-telangiectasia like 1) or Ce-rdh-1/rad-51 are more sensitive. Pachytene nuclei thus appear to effectively repair heavy ion-induced DNA damage by the meiotic homologous recombination system.

Caenorhabditis elegans Ce-rdh-1/rad-51 functions after double-strand break formation of meiotic recombination.

During meiotic prophase 1, homologous recombination is accompanied by dynamic chromosomal changes. The Ce-rdh-1/rad-51 gene is the only bacterial recA-like gene in the nematode C. elegans genome. Upon depletion of Ce-rdh-1/rad-51 using the RNA interference method, abnormal 'kinked' chromosomes can be observed in mature oocytes at diakinesis, whereas synapsis between homologous chromosomes during the pachytene stage is normal. Following fertilization, Ce-rdh-1/rad-51-depleted embryos die early in embryogenesis, and their nuclei exhibit abnormal chromosome fragments and bridges. From epistasis analyses with Ce-spo-11 defective mutant and ionizing radiation, it is indicated that Ce-rdh-1/rad-51 functions after double-strand break (DSB) formation of meiotic recombination. Under the Ce-chk-2 defective condition, whose meiotic synapsis and meiotic recombination between homologous chromosomes are completely inhibited, the Ce-rdh-1/rad51 is normally expressed in the gonadal cells. Moreover, it seems that exogenous DSBs in the Ce-chk-2 defective nuclei at the pachytene stage can be repaired between sister chromatids in a Ce-rdh-1/rad-51-dependent manner. These results indicate that Ce-rdh-1/rad51 functions after both endogenous and exogenous DSB formation during meiosis, but not as 'pairing centers' for meiotic synapsis.