Low-dose radiation induces unstable gene expression in developing human iPSC-derived retinal ganglion organoids.

The effects of low-dose radiation on undifferentiated cells carry important implications. However, the effects on developing retinal cells remain unclear. Here, we analyzed the gene expression characteristics of neuronal organoids containing immature human retinal cells under low-dose radiation and predicted their changes. Developing retinal cells generated from human induced pluripotent stem cells (iPSCs) were irradiated with either 30 or 180 mGy on days 4-5 of development for 24 h. Genome-wide gene expression was observed until day 35. A knowledge-based pathway analysis algorithm revealed fluctuations in Rho signaling and many other pathways. After a month, the levels of an essential transcription factor of eye development, the proportion of paired box 6 (PAX6)-positive cells, and the proportion of retinal ganglion cell (RGC)-specific transcription factor POU class 4 homeobox 2 (POU4F2)-positive cells increased with 30 mGy of irradiation. In contrast, they decreased after 180 mGy of irradiation. Activation of the "development of neurons" pathway after 180 mGy indicated the dedifferentiation and development of other neural cells. Fluctuating effects after low-dose radiation exposure suggest that developing retinal cells employ hormesis and dedifferentiation mechanisms in response to stress.

Analysis of inflammatory mediators in the vitreous humor of eyes with pan-uveitis according to aetiological classification.

Treatment of uveitis is complicated because of its multiple aetiologies and elevation of various inflammatory mediators. To determine the mediators that are elevated in the vitreous humor according to the aetiology of the uveitis, we examined the concentrations of 21 inflammatory cytokines, 7 chemokines, and 5 colony-stimulating/growth factors in vitreous samples from 57 eyes with uveitis associated with intraocular lymphoma (IOL, n = 13), sarcoidosis (n = 15), acute retinal necrosis (ARN, n = 13), or bacterial endophthalmitis (BE, n = 16). Samples from eyes with idiopathic epiretinal membrane (n = 15), which is not associated with uveitis, were examined as controls. Heat map analysis demonstrated that the patterns of inflammatory mediators in the vitreous humor in eyes with uveitis were disease-specific. Pairwise comparisons between the 5 diseases showed specific elevation of interferon-α2 in ARN and interleukin (IL)-6, IL-17A, and granulocyte-colony stimulating factor in BE. Pairwise comparisons between IOL, ARN, and BE revealed that levels of IL-10 in IOL, RANTES (regulated on activation, normal T cell expressed and secreted) in ARN, and IL-22 in BE were significantly higher than those in the other 2 types of uveitis. These mediators are likely to be involved in the immunopathology of specific types of uveitis and may be useful biomarkers.

Rho-Associated Coiled-Coil Kinase (ROCK) in Molecular Regulation of Angiogenesis.

Identified as a major downstream effector of the small GTPase RhoA, Rho-associated coiled-coil kinase (ROCK) is a versatile regulator of multiple cellular processes. Angiogenesis, the process of generating new capillaries from the pre-existing ones, is required for the development of various diseases such as cancer, diabetes and rheumatoid arthritis. Recently, ROCK has attracted attention for its crucial role in angiogenesis, making it a promising target for new therapeutic approaches. In this review, we summarize recent advances in understanding the role of ROCK signaling in regulating the permeability, migration, proliferation and tubulogenesis of endothelial cells (ECs), as well as its functions in non-ECs which constitute the pro-angiogenic microenvironment. The therapeutic potential of ROCK inhibitors in angiogenesis-related diseases is also discussed.

Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells.

The effects of chronic low-dose radiation on human health have not been well established. Recent studies have revealed that neural progenitor cells are present not only in the fetal brain but also in the adult brain. Since immature cells are generally more radiosensitive, here we investigated the effects of chronic low-dose radiation on cultured human neural progenitor cells (hNPCs) derived from embryonic stem cells. Radiation at low doses of 31, 124 and 496 mGy per 72 h was administered to hNPCs. The effects were estimated by gene expression profiling with microarray analysis as well as morphological analysis. Gene expression was dose-dependently changed by radiation. By thirty-one mGy of radiation, inflammatory pathways involving interferon signaling and cell junctions were altered. DNA repair and cell adhesion molecules were affected by 124 mGy of radiation while DNA synthesis, apoptosis, metabolism, and neural differentiation were all affected by 496 mGy of radiation. These in vitro results suggest that 496 mGy radiation affects the development of neuronal progenitor cells while altered gene expression was observed at a radiation dose lower than 100 mGy. This study would contribute to the elucidation of the clinical and subclinical phenotypes of impaired neuronal development induced by chronic low-dose radiation.

Rad54B serves as a scaffold in the DNA damage response that limits checkpoint strength.

The strength of the DNA damage checkpoint critically influences cell fate, yet the mechanisms behind the fine tuning of checkpoint strength during the DNA damage response (DDR) are poorly understood. Here we show that Rad54B--a SNF2 helicase-like DNA-repair protein--limits the strength of both the G1/S and G2/M checkpoints. We find that Rad54B functions as a scaffold for p53 degradation via its direct interaction with the MDM2-MDMX ubiquitin-ligase complex. During the early phases of the DDR, Rad54B is upregulated, thereby maintaining low checkpoint strength and facilitating cell cycle progression. Once the p53-mediated checkpoint is established, Rad54B is downregulated, and high checkpoint strength is maintained. Constitutive upregulation of Rad54B activity, which is frequently observed in tumours, promotes genomic instability because of checkpoint override. Thus, the scaffolding function of Rad54B dynamically regulates the maintenance of genome integrity by limiting checkpoint strength.

The ATR-Chk1 pathway plays a role in the generation of centrosome aberrations induced by Rad51C dysfunction.

Rad51C is a central component of two complexes formed by five Rad51 paralogs in vertebrates. These complexes are involved in repairing DNA double-strand breaks through homologous recombination. Despite accumulating evidence suggesting that the paralogs may prevent aneuploidy by controlling centrosome integrity, Rad51C's role in maintaining chromosome stability remains unclear. Here we demonstrate that Rad51C deficiency leads to both centrosome aberrations in an ATR-Chk1-dependent manner and increased aneuploidy in human cells. While it was reported that Rad51C deficiency did not cause centrosome aberrations in interphase in hamster cells, such aberrations were observed in interphase in HCT116 cells with Rad51C dysfunction. Caffeine treatment and down-regulation of ATR, but not that of ATM, reduced the frequency of centrosome aberrations in the mutant cells. Silencing of Rad51C by RNA interference in HT1080 cells resulted in similar aberrations. Treatment with a Chk1 inhibitor and silencing of Chk1 also reduced the frequency in HCT116 mutants. Accumulation of Chk1 at the centrosome and nuclear foci of gamma H2AX were increased in the mutants. Moreover, the mutant cells had a higher frequency of aneuploidy. These findings indicate that the ATR-Chk1 pathway plays a role in increased centrosome aberrations induced by Rad51C dysfunction.

Functional evidence for Eme1 as a marker of cisplatin resistance.

The ability to predict cisplatin sensitivity in tumors has been expected to greatly improve the outcome of cancer therapy, because the drug is frequently used in a variety of tumors. Although ERCC1 and other repair proteins have been investigated as markers of cisplatin resistance, reliable markers are still needed. Here, we demonstrate that Eme1 levels can predict cisplatin sensitivity more accurately than ERCC1 or Rad51 levels in a variety of human cancer cell lines. Eme1 forms a heterodimeric protein complex with Mus81 and functions as a structure-specific endonuclease. Haploinsufficiency of Eme1 led to hypersensitivity to cisplatin in the colon cancer cell line HCT116. On the basis of this finding, we examined the relationships between levels of proteins involved in the repair of interstrand cross-links and cisplatin sensitivity in human tumor cell lines with a variety of origins. Although ERCC1, Rad51 and Mus81 levels correlated with sensitivity to some extent, the clearest correlation was observed with Eme1. Tumors with low Eme1 levels were more sensitive to the drug than tumors with high levels. This suggests that the measurement of Eme1 in tumors may be more informative for cisplatin-based chemotherapy than that of the currently available markers.

Bloom's syndrome helicase and Mus81 are required to induce transient double-strand DNA breaks in response to DNA replication stress.

Perturbed DNA replication either activates a cell cycle checkpoint, which halts DNA replication, or decreases the rate of DNA synthesis without activating a checkpoint. Here we report that at low doses, replication inhibitors did not activate a cell cycle checkpoint, but they did activate a process that required functional Bloom's syndrome-associated (BLM) helicase, Mus81 nuclease and ataxia telangiectasia mutated and Rad3-related (ATR) kinase to induce transient double-stranded DNA breaks. The induction of transient DNA breaks was accompanied by dissociation of proliferating cell nuclear antigen (PCNA) and DNA polymerase alpha from replication forks. In cells with functional BLM, Mus81 and ATR, the transient breaks were promptly repaired and DNA continued to replicate at a slow pace in the presence of replication inhibitors. In cells that lacked BLM, Mus81, or ATR, transient breaks did not form, DNA replication did not resume, and exposure to low doses of replication inhibitors was toxic. These observations suggest that BLM helicase, ATR kinase, and Mus81 nuclease are required to convert perturbed replication forks to DNA breaks when cells encounter conditions that decelerate DNA replication, thereby leading to the rapid repair of those breaks and resumption of DNA replication without incurring DNA damage and without activating a cell cycle checkpoint.

Haploinsufficiency of RAD51B causes centrosome fragmentation and aneuploidy in human cells.

The Rad51-like proteins, Rad51B, Rad51C, Rad51D, XRCC2, and XRCC3, have been shown to form two distinct complexes and seem to assist Rad51 in the early stages of homologous recombination. Although these proteins share sequence similarity with Rad51, they do not show functional redundancy. Among them, Rad51B is unique in that the gene maps to the human chromosome 14q23-24, the region frequently involved in balanced chromosome translocations in benign tumors particularly in uterine leiomyomas. Despite accumulating descriptive evidence of altered Rad51B function in these tumors, the biological significance of this aberration is still unknown. To assess the significance of reduced Rad51B function, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here, we show that haploinsufficiency of RAD51B causes mild hypersensitivity to DNA-damaging agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation, and an increase in chromosome aberrations. Remarkably, haploinsufficiency of RAD51B leads to centrosome fragmentation and aneuploidy. In addition, an approximately 50% reduction in RAD51B mRNA levels by RNA interference also leads to centrosome fragmentation in the human fibrosarcoma cell line HT1080. These findings suggest that the proper biallelic expression of RAD51B is required for the maintenance of chromosome integrity in human cells.

Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells.

The Mus81-Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3'-flap structures and aberrant replication fork structures. Mus81-/- mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G2/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 or Eme1 also causes similar phenotypes. These findings suggest that a complex network of the checkpoint pathways that respond to DNA double-strand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency.

XRCC3 deficiency results in a defect in recombination and increased endoreduplication in human cells.

XRCC3 was inactivated in human cells by gene targeting. Consistent with its role in homologous recombination, XRCC3(-/-) cells showed a two-fold sensitivity to DNA cross-linking agents, a mild reduction in sister chromatid exchange, impaired Rad51 focus formation and elevated chromosome aberrations. Furthermore, endoreduplication was increased five- seven-fold in the mutants. The T241M variant of XRCC3 has been associated with an increased cancer risk. Expression of the wild-type cDNA restored this phenotype, while expression of the variant restored the defective recombinational repair, but not the increased endoreduplication. RPA, a protein essential for homologous recombination and DNA replication, is associated with XRCC3 and Rad52. Overexpression of RPA promoted endoreduplication, which was partially complemented by overexpression of the wild-type XRCC3 protein, but not by overexpression of the variant protein. Overexpression of Rad52 prevented endoreduplication in RPA-overexpressing cells, in XRCC3(-/-) cells and in the variant-expressing cells, suggesting that deregulated RPA was responsible for the increased endoreduplication. These observations offer the first genetic evidence for the association between homologous recombination and replication initiation having a role in cancer susceptibility.

Retinal pigment epithelial tear involving the fovea with preserved visual function.

Tears of the retinal pigment epithelium are known to occur either spontaneously or after laser photocoagulation in eyes with retinal pigment epithelium detachment. A 65-year-old man with preexisting retinal pigment epithelium detachment developed a retinal pigment epithelium tear after dye laser retinal photocoagulation. The tear gradually expanded to involve the fovea, but his best-corrected visual acuity remained 0.7 in the left eye during 20 months. Optical coherence tomography showed a defect of the retinal pigment epithelium with absence of regeneration. Scanning laser ophthalmoscopy revealed his fixation approached intact retinal pigment epithelium, but was still beneath the fovea. This case may indicate that the retinal pigment epithelium directly beneath the central macula is not essential for maintenance of the overlying foveal function under some conditions.

A role for RAD54B in homologous recombination in human cells.

In human somatic cells, homologous recombination is a rare event. To facilitate the targeted modification of the genome for research and gene therapy applications, efforts should be directed toward understanding the molecular mechanisms of homologous recombination in human cells. Although human genes homologous to members of the RAD52 epistasis group in yeast have been identified, no genes have been demonstrated to play a role in homologous recombination in human cells. Here, we report that RAD54B plays a critical role in targeted integration in human cells. Inactivation of RAD54B in a colon cancer cell line resulted in severe reduction of targeted integration frequency. Sensitivity to DNA-damaging agents and sister-chromatid exchange were not affected in RAD54B-deficient cells. Parts of these phenotypes were similar to those of Saccharomyces cerevisiae tid1/rdh54 mutants, suggesting that RAD54B may be a human homolog of TID1/RDH54. In yeast, TID1/RDH54 acts in the recombinational repair pathway via roles partially overlapping those of RAD54. Our findings provide the first genetic evidence that the mitotic recombination pathway is functionally conserved from yeast to humans.