Genotoxic stress causes proliferating cells to activate the DNA damage checkpoint, to assist DNA damage recovery by slowing cell cycle progression. Thus, to drive proliferation, cells must tolerate DNA damage and suppress the checkpoint response. However, the mechanism underlying this negative regulation of checkpoint activation is still elusive. We show that human Cyclin-Dependent-Kinases (CDKs) target the RAD9 subunit of the 9-1-1 checkpoint clamp on Thr292, to modulate DNA damage checkpoint activation. Thr292 phosphorylation on RAD9 creates a binding site for Polo-Like-Kinase1 (PLK1), which phosphorylates RAD9 on Thr313. These CDK-PLK1-dependent phosphorylations of RAD9 suppress checkpoint activation, therefore maintaining high DNA synthesis rates during DNA replication stress. Our results suggest that CDK locally initiates a PLK1-dependent signaling response that antagonizes the ability of the DNA damage checkpoint to detect DNA damage. These findings provide a mechanism for the suppression of DNA damage checkpoint signaling, to promote cell proliferation under genotoxic stress conditions.

Multiple epigenetic pathways underlie the temporal order of DNA replication (replication timing) in the contexts of development and disease. DNA methylation by DNA methyltransferases (Dnmts) and downstream chromatin reorganization and transcriptional changes are thought to impact DNA replication, yet this remains to be comprehensively tested. Using cell-based and genome-wide approaches to measure replication timing, we identified a number of genomic regions undergoing subtle but reproducible replication timing changes in various Dnmt-mutant mouse embryonic stem (ES) cell lines that included a cell line with a drug-inducible Dnmt3a2 expression system. Replication timing within pericentromeric heterochromatin (PH) was shown to be correlated with redistribution of H3K27me3 induced by DNA hypomethylation: Later replicating PH coincided with H3K27me3-enriched regions. In contrast, this relationship with H3K27me3 was not evident within chromosomal arm regions undergoing either early-to-late (EtoL) or late-to-early (LtoE) switching of replication timing upon loss of the Dnmts. Interestingly, Dnmt-sensitive transcriptional up- and downregulation frequently coincided with earlier and later shifts in replication timing of the chromosomal arm regions, respectively. Our study revealed the previously unrecognized complex and diverse effects of the Dnmts loss on the mammalian DNA replication landscape.

Osteoclasts are multinucleated cells formed by macrophage cell fusion that are responsible for bone resorption. Previously, we found that treating osteoclastic progenitor cells with (−)-epigallocatechin gallate (EGCg) increased cell fusion. In this study, we aimed to identify factors involved in the cell fusion induced by EGCg. We hypothesized that EGCg-induced oxidative stress might be involved in cell fusion, and used macrophage cell line RAW264.7 cells. We evaluated cell fusion activity after adding the antioxidants N-acetyl-l-cysteine (NAC) or catalase in addition to EGCg. The mRNA expressions of genes related to cell fusion and bone resorption were quantified by real-time PCR. Finally, we added hydrogen peroxide and examined its effects on cell fusion and TRAP activity. EGCg-induced cell fusion was strongly inhibited by the addition of NAC in a dose-dependent manner (EGCg with 5 mM NAC; decreased to 1.5%; p < 0.05), while the inhibitory effect of catalase was limited (EGCg with 500 U/mL catalase; decreased to 27.7%; p < 0.05). DC-STAMP expression was significantly upregulated by EGCg compared with the untreated group, and the upregulation was significantly suppressed by 5 mM NAC. Conversely, Nfatc1 and TRAP expression were not upregulated by EGCg. These results suggest that EGCg induces DC-STAMP expression via reactive oxygen species production, which regulates cell fusion but does not affect the osteoclastic pathway. Although treatment with hydrogen peroxide promoted the formation of multinucleated cells, no increase in TRAP activity was observed, which was similar to EGCg treatment. This study suggests that the increased cell fusion by EGCg may be induced by oxidative stress due to reactive oxygen species production.

Background/Purpose: Some health-beneficial foods, including adzuki beans ( Vigna angularis), can show antihyperglycemic effects. Adzuki bean-derived polyphenols, such as (+)-catechin 7- O- β-d-glucopyranoside (C7G) and (+)-epicatechin 7- O- β-d-glucopyranoside (E7G), could be potential inhibitors of α-amylase and α-glucosidase, however, few studies of the activity of adzuki polyphenols have been performed in vivo. Thus, we evaluated the antihyperglycemic effects of crude adzuki bean extract containing polyphenols (ABP), and purified C7G or E7G using mice and Caco-2 cells. Methods: Six-week-old male ICR mice were orally administered ABP (100 or 250 mg/kg), C7G or E7G (15 or 40 mg/kg), and sucrose (2 g/kg). Blood samples were taken from the lateral tail vein to measure the blood glucose levels at various times over 0–120 min. Cultured caco-2 cells, an intestinal model, were treated with ABP, C7G, or E7G. The glucose concentration in the medium was measured to examine the activity of α-glucosidase. Results: Administration of adzuki polyphenols decreased blood glucose levels, especially E7G administration significantly showed antihyperglycemic effects in vivo. In Caco-2 cells, α-glucosidase activity was significantly decreased by ABP, C7G, or E7G addition in a dose-dependent manner without cytotoxicity. Conclusion: These results suggest that C7G and E7G in adzuki bean extract inhibited sucrose-degrading enzymes to elicit antihyperglycemic effects in vivo, which supports the results of previous studies using adzuki beans, and may contribute to health promotion.

L-theanine is derived from green tea leaves. It has neuroprotective properties that promote brain development through the migration of cerebellar granule cells, but its exact mechanisms remain unclear. In this study, we examine receptor-mediated CGC migration signaling pathways modulated by l-theanine. We treated cerebellar explants and primary CGCs from postnatal mice with l-theanine (0.1-1000 µm), with and without receptor blockers CNQX, MK-801, and bicuculline. Changes at the cellular level and activation of signaling proteins were studied. The results showed that l-theanine stimulated CGC migration in a dose- and time-dependent manner. This stimulatory effect was nullified by blocking the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid receptors and was enhanced by blocking GABAA receptors. Moreover, l-theanine stimulated the phosphorylation of ERK1/2, calmodulin kinase II, CREB, and AMPA, indicating activation of pathways related to synaptic plasticity and cytoskeletal changes. These findings suggest that l-theanine likely promotes CGC migration through specific receptor pathways, supporting its role in neurogenesis.