VEGF affects mitochondrial ROS generation in glioma cells and acts as a radioresistance factor.

Vascular endothelial growth factor (VEGF) is closely related to angiogenesis. Anticancer therapy by inhibiting VEGF signaling is well established. However, the role of VEGF in cell-cell communication during the response to ionizing radiation is not well understood. Here, we examined the role of VEGF on radiosensitivity of cells. The addition of recombinant VEGF (rVEGF) on cultured rat C6 glioma cells showed a radioprotective effects on X-ray irradiation and reduced oxidative stress. These effects were also observed by endogenous VEGF in supernatant of C6 glioma cells. Reduction of oxidative stress by VEGF is suggested to underlie the radioprotective effects. The mechanism of VEGF-induced reduction of oxidative stress was indicated by a decreased oxygen consumption rate (OCR) in mitochondria. However, the number of DNA double-strand breaks (DSB) immediately after irradiation was not reduced by the treatment with VEGF. These results suggest that VEGF plays a role in cell survival after irradiation by controlling the oxidative condition through mitochondrial function that is independent of the efficiency of DSB induction.

C9orf72-Derived Proline:Arginine Poly-Dipeptides Modulate Cytoskeleton and Mechanical Stress Response.

Proline:arginine (PR) poly-dipeptides from the GGGGCC repeat expansion in C9orf72 have cytotoxicity and bind intermediate filaments (IFs). However, it remains unknown how PR poly-dipeptides affect cytoskeletal organization and focal adhesion (FA) formation. Here, we show that changes to the cytoskeleton and FA by PR poly-dipeptides result in the alteration of cell stiffness and mechanical stress response. PR poly-dipeptides increased the junctions and branches of the IF network and increased cell stiffness. They also changed the distribution of actin filaments and increased the size of FA and intracellular calcium concentration. PR poly-dipeptides or an inhibitor of IF organization prevented cell detachment. Furthermore, PR poly-dipeptides induced upregulation of mechanical stress response factors and led to a maladaptive response to cyclic stretch. These results suggest that the effects of PR poly-dipeptides on mechanical properties and mechanical stress response may serve as a pathogenesis of C9orf72-related neurodegeneration.

Gene Expression Profiles of Human Cerebral Organoids Identify PPAR Pathway and PKM2 as Key Markers for Oxygen-Glucose Deprivation and Reoxygenation.

Ischemic stroke is one of the most common neurological diseases. However, the impact of ischemic stroke on human cerebral tissue remains largely unknown due to a lack of ischemic human brain samples. In this study, we applied cerebral organoids derived from human induced pluripotent stem cells to evaluate the effect of oxygen-glucose deprivation/reoxygenation (OGD/R). Pathway analysis showed the relationships between vitamin digestion and absorption, fat digestion and absorption, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and complement and coagulation cascades. Combinational verification with transcriptome and gene expression analysis of different cell types revealed fatty acids-related PPAR signaling pathway and pyruvate kinase isoform M2 (PKM2) as key markers of neuronal cells in response to OGD/R. These findings suggest that, although there remain some limitations to be improved, our ischemic stroke model using human cerebral organoids would be a potentially useful tool when combined with other conventional two-dimensional (2D) mono-culture systems.

27-Hydroxycholesterol regulates human SLC22A12 gene expression through estrogen receptor action.

The excretion and reabsorption of uric acid both to and from urine are tightly regulated by uric acid transporters. Metabolic syndrome conditions, such as obesity, hypercholesterolemia, and insulin resistance, are believed to regulate the expression of uric acid transporters and decrease the excretion of uric acid. However, the mechanisms driving cholesterol impacts on uric acid transporters have been unknown. Here, we show that cholesterol metabolite 27-hydroxycholesterol (27HC) upregulates the uric acid reabsorption transporter URAT1 encoded by SLC22A12 via estrogen receptors (ER). Transcriptional motif analysis showed that the SLC22A12 gene promoter has more estrogen response elements (EREs) than other uric acid reabsorption transporters such as SLC22A11 and SLC22A13, and 27HC-activated SLC22A12 gene promoter via ER through EREs. Furthermore, 27HC increased SLC22A12 gene expression in human kidney organoids. Our results suggest that in hypercholesterolemic conditions, elevated levels of 27HC derived from cholesterol induce URAT1/SLC22A12 expression to increase uric acid reabsorption, and thereby, could increase serum uric acid levels.

Inhibition of the ATR kinase enhances 5-FU sensitivity independently of nonhomologous end-joining and homologous recombination repair pathways.

The anticancer agent 5-fluorouracil (5-FU) is cytotoxic and often used to treat various cancers. 5-FU is thought to inhibit the enzyme thymidylate synthase, which plays a role in nucleotide synthesis and has been found to induce single- and double-strand DNA breaks. ATR Ser/Thr kinase (ATR) is a principal kinase in the DNA damage response and is activated in response to UV- and chemotherapeutic drug-induced DNA replication stress, but its role in cellular responses to 5-FU is unclear. In this study, we examined the effect of ATR inhibition on 5-FU sensitivity of mammalian cells. Using immunoblotting, we found that 5-FU treatment dose-dependently induced the phosphorylation of ATR at the autophosphorylation site Thr-1989 and thereby activated its kinase. Administration of 5-FU with a specific ATR inhibitor remarkably decreased cell survival, compared with 5-FU treatment combined with other major DNA repair kinase inhibitors. Of note, the ATR inhibition enhanced induction of DNA double-strand breaks and apoptosis in 5-FU-treated cells. Using gene expression analysis, we found that 5-FU induced the activation of the intra-S cell-cycle checkpoint. Cells lacking BRCA2 were sensitive to 5-FU in the presence of ATR inhibitor. Moreover, ATR inhibition enhanced the efficacy of the 5-FU treatment, independently of the nonhomologous end-joining and homologous recombination repair pathways. These findings suggest that ATR could be a potential therapeutic target in 5-FU-based chemotherapy.

Brainstem Organoids From Human Pluripotent Stem Cells.

The brainstem is a posterior region of the brain, composed of three parts, midbrain, pons, and medulla oblongata. It is critical in controlling heartbeat, blood pressure, and respiration, all of which are life-sustaining functions, and therefore, damages to or disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells (hPSCs) recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models are limited in the extent hPSCs recapitulate human brain development and hence are not able to fully elucidate the diseases affecting various components of the brain such as brainstem. Here, we developed a method to generate human brainstem organoids (hBSOs), containing midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, dopaminergic neurons, and neural crest lineage cells. Single-cell RNA sequence (scRNA-seq) analysis, together with evidence from proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem, which raises the possibility of making use of hBSOs in investigating central nervous system disorders affecting brainstem and in efficient drug screenings.

Early and Delayed Induction of DSBs by Nontargeted Effects in ICR Mouse Lymphocytes after In Vivo X Irradiation.

The goal of this study was to determine whether in vivo X irradiation induces nontargeted effects, such as delayed effects and bystander effects in ICR mouse lymphocytes. We first examined the generation of DNA double-strand breaks (DSBs) in lymphocytes, isolated from ICR mice exposed to 1 Gy X irradiation, by enumeration of p53 binding protein 1 (53BP1) foci, and observed that the number of 53BP1 foci reached their maximum 3 days postirradiation and decreased to background level 30 days postirradiation. However, the number of 53BP1 foci was significantly increased in lymphocytes isolated from ICR mice 90-365 days postirradiation. This result indicates that in vivo X irradiation induced delayed DSBs in ICR mouse lymphocytes. We next counted the number of 53BP1 foci in lymphocytes isolated from sham-irradiated ICR mice that had been co-cultured with lymphocytes isolated from 1 Gy X-irradiated ICR mice, and observed a significant increase in the number of 53BP1 foci 1-7 days postirradiation. This result indicates that in vivo X irradiation induced bystander effects in ICR mouse lymphocytes. These findings suggest that in vivo X irradiation induces early and delayed nontargeted effects in ICR mouse lymphocytes.

Gemcitabine Induces Radiosensitization Through Inhibition of RAD51-dependent Repair for DNA Double-strand Breaks.

BACKGROUND/AIM: Gemcitabine (GEM) is used in clinical chemo-radiotherapy; however, the mechanism that contributes to enhanced radiosensitivity by GEM is not fully-understood. We evaluated the effect of GEM on radiosensitization in pancreatic cancer cell lines. MATERIALS AND METHODS: Pancreatic cell lines PK-59 and PK-45p were used. A total of 5 µM GEM for 4 h were administered pre- or post-gamma irradiation. RESULTS: Enhanced cell killing effects by GEM in radiotherapy were observed for pre-treatment but not post-treatment GEM. We focused on the dynamics of RAD51 and phospho-H2AX foci after irradiation. Significantly higher numbers of phospho-H2AX foci were observed in GEM pre-treated cells than in untreated cells after irradiation. We also found inhibition of the formation and degradation of RAD51 foci by GEM pre-treatment. The radiosensitizing effect of GEM was suppressed by knockdown of RAD51. CONCLUSION: RAD51-dependent homologous recombination is one of the key targets in the GEM-induced radiosensitizing effect.

Relief of delayed oxidative stress by ascorbic acid can suppress radiation-induced cellular senescence in mammalian fibroblast cells.

Ionizing radiation-induced cellular senescence is thought to be caused by nuclear DNA damage that cannot be repaired. However, here we found that radiation induces delayed increase of intracellular oxidative stress after irradiation. We investigated whether the relief of delayed oxidative stress by ascorbic acid would suppress the radiation-induced cellular senescence in Syrian golden hamster embryo (SHE) cells. We observed that the level of oxidative stress was drastically increased soon after irradiation, then declined to the level in non-irradiated cells, and increased again with a peak on day 3 after irradiation. We found that the inductions of cellular senescence after X-irradiation were reduced along with suppression of the delayed induction of oxidative stress by treatment with ascorbic acid, but not when oxidative stress occurred immediately after irradiation. Moreover, treatment of ascorbic acid inhibited p53 accumulation at 3 days after irradiation. Our data suggested a delayed increase of intracellular oxidative stress levels plays an important role in the process of radiation-induced cellular senescence by p53 accumulation.

Ionizing radiation-induced cell death is partly caused by increase of mitochondrial reactive oxygen species in normal human fibroblast cells.

Radiation-induced cell death is thought to be caused by nuclear DNA damage that cannot be repaired. However, in this study we found that a delayed increase of mitochondrial reactive oxygen species (ROS) is responsible for some of the radiation-induced cell death in normal human fibroblast cells. We have previously reported that there is a delayed increase of mitochondrial (·)O2(-), measured using MitoSOX™ Red reagent, due to gamma irradiation. This is dependent on Drp1 localization to mitochondria. Here, we show that knockdown of Drp1 expression reduces the level of DNA double-strand breaks (DSBs) remaining 3 days after 6 Gy irradiation. Furthermore, cells with knockdown of Drp1 expression are more resistant to gamma radiation. We then tested whether the delayed increase of ROS causes DNA damage. The antioxidant, 2-glucopyranoside ascorbic acid (AA-2G), was applied before or after irradiation to inhibit ROS production during irradiation or to inhibit delayed ROS production from mitochondria. Interestingly, 1 h after exposure, the AA-2G treatment reduced the level of DSBs remaining 3 days after 6 Gy irradiation. In addition, irradiated AA-2G-treated cells were more resistant to radiation than the untreated cells. These results indicate that delayed mitochondrial ROS production may cause some of the cell death after irradiation.

Comparison of the biological effects of (18)F at different intracellular levels.

We herein examined the biological effects of cells treated with (18)F labeled drugs for positron emission tomography (PET). The relationship between the intracellular distribution of (18)F and levels of damaged DNA has yet to be clarified in detail. We used culture cells (Chinese Hamster Ovary cells) treated with two types of (18)F labeled drugs, fluorodeoxyglucose (FDG) and fluorine ion (HF). FDG efficiently accumulated in cells, whereas HF did not. To examine the induction of DNA double strand breaks (DSB), we measured the number of foci for 53BP1 that formed at the site of DNA DSB. The results revealed that although radioactivity levels were the same, the induction of 53BP1 foci was stronger in cells treated with (18)F-FDG than in those treated with (18)F-HF. The clonogenic survival of cells was significantly lower with (18)F-FDG than with (18)F-HF. We concluded that the efficient accumulation of (18)F in cells led to stronger biological effects due to more severe cellular lethality via the induction of DNA DSB.

Ionizing radiation accelerates Drp1-dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells.

Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial O(2)(-) production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial O(2)(-). Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.

Epigenetic gene silencing is a novel mechanism involved in delayed manifestation of radiation-induced genomic instability in mammalian cells.

We examined mechanisms involved in delayed mutagenesis in CHO-LacZeo cells harboring the fusion gene between the bacterial LacZ and the Zeocin-resistance genes. After X irradiation, Zeocin-resistant primary colonies were isolated, and the primary clones were subjected to the secondary colony formation in the absence of Zeocin. We found that the surviving primary clones showed a significantly higher delayed mutation frequency compared with those derived from nonirradiated CHO-LacZeo cells. The mutation spectrum of the LacZ gene was analyzed by the LacZ gene-specific PCR. We found that more than 90% of the spontaneous and direct mutants were PCR-product negative, indicating that deletion of the LacZ gene was a predominant change in these mutants. While deletion of the LacZ gene was also observed in delayed mutants, we found that more than 20% of delayed mutants had a PCR product similar to that of the parental CHO-LacZeo cells. These PCR product-positive mutants spontaneously reverted to LacZ-positive (LacZ(+)) cells, and all of these mutants became LacZ-positive after 5-azacytidine treatment. These results indicate that epigenetic gene silencing, in addition to elevated recombination, is involved in delayed mutagenesis, which is a novel mechanism underlying delayed manifestations of radiation-induced genomic instability.