Eiichiro Mori

The Oxygen-Rich Postnatal Environment Induces Cardiomyocyte Cell-Cycle Arrest through DNA Damage Response

The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches.

Evidence for the Involvement of Double-Strand Breaks in Heat-Induced Cell Killing

To identify critical events associated with heat-induced cell killing, we examined foci formation of γH2AX (histone H2AX phosphorylated at serine 139) in heat-treated cells. This assay is known to be quite sensitive and a specific indicator for the presence of double-strand breaks. We found that the number of γH2AX foci increased rapidly and reached a maximum 30 minutes after heat treatment, as well as after X-ray irradiation. When cells were heated at 41.5°C to 45.5°C, we observed a linear increase with time in the number of γH2AX foci. An inflection point at 42.5°C and the thermal activation energies above and below the inflection point were almost the same for cell killing and foci formation according to Arrhenius plot analysis. From these results, it is suggested that the number of γH2AX foci is correlated with the temperature dependence of cell killing. During periods when cells were exposed to heat, the cell cycle-dependent pattern of cell killing was the same as the cell cycle pattern of γH2AX foci formation. We also found that thermotolerance was due to a depression in the number of γH2AX foci formed after heating when the cells were pre-treated by heat. These findings suggest that cell killing might be associated with double-strand break formation via protein denaturation.

DNA double-strand breaks: Their production, recognition, and repair in eukaryotes

Human cells accumulate at least 10,000 DNA lesions every day. Failure to repair such lesions can lead to mutations, genomic instability, or cell death. Among the various types of damage which can be expressed in a cell, DNA double-strand breaks (DSBs) represent the most serious threat. Different kinds of physical, chemical, and biological factors have been reported to induce DNA lesions, including DSBs. The aim of this review is to provide a basic understanding and overview of how DSBs are produced, recognized and repaired, and to describe the role of some of the genes and proteins involved in DSB repair.

A Method for Systematic Mapping of Protein Lysine Methylation Identifies Functions for HP1β in DNA Damage Response

Lysine methylation occurs on both histone and nonhistone proteins. However, our knowledge on the prevalence and function of nonhistone protein methylation is poor. We describe an approach that combines peptide array, bioinformatics, and mass spectrometry to systematically identify lysine methylation sites and map methyllysine-driven protein-protein interactions. Using this approach, we identified a high-confidence and high-resolution interactome of the heterochromatin protein 1β (HP1β) and uncovered, simultaneously, numerous methyllysine sites on nonhistone proteins. We found that HP1β binds to DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and regulates its localization to double-strand breaks (DSBs) during DNA damage response (DDR). Mutation of the methylation sites in DNA-PKcs or depletion of HP1β in cells caused defects in DDR. Furthermore, we showed that the methylation of DNA-PKcs and many other proteins in the HP1β interactome undergoes large changes in response to DNA damage, indicating that Lys methylation is a highly dynamic posttranslational modification.

Toxic PRn poly-dipeptides encoded by the C9orf72 repeat expansion block nuclear import and export

Significance A hexanucleotide repeat expansion in the first intron of the C9orf72 gene represents the most prominent form of heritable amyotrophic lateral sclerosis. Bidirectional transcription and ATG-independent translation of the expanded (GGGGCC)n repeat specifies the production of toxic glycine:arginine (GRn) and proline:arginine (PRn) poly-dipeptides. The present study provides evidence that the PRn poly-dipeptide binds directly to the central channel of nuclear pores, causing inhibition of both the import and export of macromolecules to and from the nucleus. Nuclear pore binding is shown to be mediated via direct interaction between the toxic PRn poly-dipeptide and polymeric forms of nuclear pore proteins enriched in phenylalanine:glycine repeats. The toxic proline:arginine (PRn) poly-dipeptide encoded by the (GGGGCC)n repeat expansion in the C9orf72 form of heritable amyotrophic lateral sclerosis (ALS) binds to the central channel of the nuclear pore and inhibits the movement of macromolecules into and out of the nucleus. The PRn poly-dipeptide binds to polymeric forms of the phenylalanine:glycine (FG) repeat domain, which is shared by several proteins of the nuclear pore complex, including those in the central channel. A method of chemical footprinting was used to characterize labile, cross-β polymers formed from the FG domain of the Nup54 protein. Mutations within the footprinted region of Nup54 polymers blocked both polymerization and binding by the PRn poly-dipeptide. The aliphatic alcohol 1,6-hexanediol melted FG domain polymers in vitro and reversed PRn-mediated enhancement of the nuclear pore permeability barrier. These data suggest that toxicity of the PRn poly-dipeptide results in part from its ability to lock the FG repeats of nuclear pore proteins in the polymerized state. Our study offers a mechanistic interpretation of PRn poly-dipeptide toxicity in the context of a prominent form of ALS.

Heat induces γH2AX foci formation in mammalian cells

H2AX is a histone variant which is present and ubiquitously distributed throughout the genome. An immunocytochemical assay using antibodies capable of recognizing histone H2AX phosphorylated at serine 139 (gammaH2AX) is very sensitive and is a specific indicator for the existence of a DNA double strand break. Although heat stress has been reported to induce the formation of gammaH2AX foci, no gammaH2AX foci formation was observed in several mammalian cell lines after heat shock. Since this was in contrast to earlier reports, the work described here was intended to verify that heat-induced gammaH2AX foci do form in mammalian cell lines other than the cell lines used in earlier reports concerning gammaH2AX foci formation. The cell lines used in this work includes cell lines with differing p53-gene status (H1299, H1299/neo, H1299/mp53 and H1299/wtp53 cells), various cancer cell lines (HeLa, HepG2, U2-OS cells), normal human cells (HEK-293 and AG1522), and cell lines established from other species (MEF normal mouse cells and CHL normal Chinese hamster cells). Exponentially growing cells were exposed to heat shock (42 degrees C for 6 h or 45.5 degrees C for 20 min) or to X-rays (3Gy). The presence of gammaH2AX was examined with immunocytochemistry and flow cytometry. Induction of gammaH2AX foci formation was observed in all of the mammalian cell lines used here after heat-treatment as well as after X-irradiation. However, the intensity of gammaH2AX was different in the different cell lines used. These results confirm that heat can induce gammaH2AX foci formation in many mammalian cell lines.

High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation.

Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation‐induced apoptosis. Human gingival cancer cells (Ca9‐22 cells) containing a mutated p53 (mp53) gene were irradiated with X‐rays, C‐ion (13–100 KeV/µm), or Fe‐ion beams (200 KeV/µm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase‐3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET‐dependent manner. Both RBE curves reached a peak at 100 KeV/µm, and then decreased at values over 100 KeV/µm. When cells were irradiated with high LET radiation, Caspase‐3 was cleaved and activated, leading to poly (ADP‐ribose) polymerase (PARP) cleavage. In addition, Caspase‐9 inhibitor suppressed Caspase‐3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase‐8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase‐3 through Caspase‐9, even in the presence of mp53. (Cancer Sci 2008; 99: 1455–1460)

Development of thermotolerance requires interaction between polymerase-β and heat shock proteins

Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat‐induced DNA double‐strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat‐labile repair proteins such as DNA polymerase‐β (Polβ). Here the authors show that thermotolerance was partially suppressed in Polβ−/– mouse embryonic fibroblasts (MEF) when compared to the wild‐type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat‐induced γH2AX was suppressed in the thermotolerant cells. These results suggest that Polβ at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat‐induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat‐labile proteins besides Polβ. The present novel findings provide strong evidence that Polβ functions as a critical element involved in thermotolerance and exerts an important role in heat‐induced DSB. (Cancer Sci 2008; 99: 973–978)

Nonenzymatic Role for WRN in Preserving Nascent DNA Strands after Replication Stress

WRN, the protein defective in Werner syndrome (WS), is a multifunctional nuclease involved in DNA damage repair, replication, and genome stability maintenance. It was assumed that the nuclease activities of WRN were critical for these functions. Here, we report a nonenzymatic role for WRN in preserving nascent DNA strands following replication stress. We found that lack of WRN led to shortening of nascent DNA strands after replication stress. Furthermore, we discovered that the exonuclease activity of MRE11 was responsible for the shortening of newly replicated DNA in the absence of WRN. Mechanistically, the N-terminal FHA domain of NBS1 recruits WRN to replication-associated DNA double-stranded breaks to stabilize Rad51 and to limit the nuclease activity of its C-terminal binding partner MRE11. Thus, this previously unrecognized nonenzymatic function of WRN in the stabilization of nascent DNA strands sheds light on the molecular reason for the origin of genome instability in WS individuals.

SUMOylation of ATRIP potentiates DNA damage signaling by boosting multiple protein interactions in the ATR pathway

The ATR (ATM [ataxia telangiectasia-mutated]- and Rad3-related) checkpoint is a crucial DNA damage signaling pathway. While the ATR pathway is known to transmit DNA damage signals through the ATR-Chk1 kinase cascade, whether post-translational modifications other than phosphorylation are important for this pathway remains largely unknown. Here, we show that protein SUMOylation plays a key role in the ATR pathway. ATRIP, the regulatory partner of ATR, is modified by SUMO2/3 at K234 and K289. An ATRIP mutant lacking the SUMOylation sites fails to localize to DNA damage and support ATR activation efficiently. Surprisingly, the ATRIP SUMOylation mutant is compromised in the interaction with a protein group, rather than a single protein, in the ATR pathway. Multiple ATRIP-interacting proteins, including ATR, RPA70, TopBP1, and the MRE11-RAD50-NBS1 complex, exhibit reduced binding to the ATRIP SUMOylation mutant in cells and display affinity for SUMO2 chains in vitro, suggesting that they bind not only ATRIP but also SUMO. Fusion of a SUMO2 chain to the ATRIP SUMOylation mutant enhances its interaction with the protein group and partially suppresses its localization and functional defects, revealing that ATRIP SUMOylation promotes ATR activation by providing a unique type of protein glue that boosts multiple protein interactions along the ATR pathway.