Masahiko Kobayashi

Identification of a Lipid Peroxidation Product as a Potential Trigger of the p53 Pathway

The tumor suppressor and transcription factor p53 is a key modulator of cellular stress responses, and activation of p53 can trigger apoptosis in many cell types, including neurons. We found that this nuclear protein was significantly phosphorylated when human neuroblastoma SH-SY5Y cells were exposed to in vitro oxidized polyunsaturated fatty acids. To identify an oxidized lipid that induces p53 phosphorylation, we conducted a screening of lipid peroxidation products in human neuroblastoma SH-SY5Y cells and identified 4-oxo-2-nonenal (ONE), a recently identified aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids, as a potential inducer of the p53 phosphorylation. We also found that ONE induced the phosphorylation of ataxia telangiectasia-mutated, which plays an essential role in transmitting DNA damage signals by the phosphorylation of p53. In addition, exposure of the cells to ONE resulted in an accumulation of ubiquitinated proteins and in a significant inhibition of proteasome activities, suggesting that ONE acted on the ubiquitin-proteasome pathway, a regulatory mechanism of p53 turnover. In addition, the observation that the ONE-induced p53 response was associated with the induction of apoptosis suggested that ONE activated the p53-dependent apoptosis mechanism via activation of the p53 signaling pathway and down-regulation of the p53 turnover. Finally, we observed that the ONE-2′-deoxyguanosine adduct, 7-(2-oxo-heptyl)-substituted 1,N2-etheno-2′-deoxyguanosine, was accumulated in the spinal cord motor neurons of patients with sporadic amyotrophic lateral sclerosis. These data may suggest the potential critical role for ONE in the induction of a neuronal apoptosis program during oxidative processes.

Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication

The Rad17‐replication factor C (Rad17‐RFC) and Rad9‐Rad1‐Hus1 complexes are thought to function in the early phase of cell‐cycle checkpoint control as sensors for genome damage and genome replication errors. However, genetic analysis of the functions of these complexes in vertebrates is complicated by the lethality of these gene disruptions in embryonic mouse cells. We disrupted the Rad17 and Rad9 loci by gene targeting in the chicken B lymphocyte line DT40. Rad17−/− and Rad9−/− DT40 cells are viable, and are highly sensitive to UV irradiation, alkylating agents, and DNA replication inhibitors, such as hydroxyurea. We further found that Rad17−/− and Rad9−/− but not ATM−/− cells are defective in S‐phase DNA damage checkpoint controls and in the cellular response to stalled DNA replication. These results indicate a critical role for chicken Rad17 and Rad9 in the cellular response to stalled DNA replication and DNA damage.

ATR-ATRIP kinase complex triggers activation of the fanconi anemia DNA repair pathway

ATR kinase activates the S-phase checkpoint when replication forks stall at sites of DNA damage. This event also causes phosphorylation of the Fanconi anemia (FA) protein FANCI, triggering its monoubiquitination of the key DNA repair factor FANCD2 by the FA core E3 ligase complex, thereby promoting this central pathway of DNA repair which permits replication to be restarted. However, the interplay between ATR and the FA pathway has been unclear. In this study, we present evidence that their action is directly linked, gaining insights into this relationship in a DT40 mutant cell line that is conditionally deficient in the critical ATR-binding partner protein ATRIP. Using this system, we showed that ATRIP was crucial for DNA damage-induced FANCD2 monoubiquitination and FANCI phosphorylation. ATR kinase phosphorylated recombinant FANCI protein in vitro, which was facilitated by the presence of FANCD2. Mechanistic investigations revealed that the RPA region but not the TopBP1 region of ATRIP was required for FANCD2 monoubiquitination, whereas Chk1 phosphorylation relied upon both domains. Together, our findings identify ATR as the kinase responsible for activating the FA pathway of DNA repair.

Lysosome is a primary organelle in B cell receptor-mediated apoptosis: An indispensable role of Syk in lysosomal function

To investigate the mechanism of B cell receptor (BCR)‐mediated apoptosis, we utilized immature B cell lines, DT40 and WEHI‐231. In both cell lines, BCR‐crosslinking caused the increase in lysosomal pH with early apoptotic changes characterized by chromatin condensation and phosphatidylserine exposure. This increase was detected in c‐Abl‐deficient DT40 cells but not in Syk‐deficient cells, which corresponded to the fact that the former cells but not the latter revealed BCR‐induced apoptosis. In contrast, BCR‐crosslinking caused no apparent change in mitochondrial transmembrane potential. Therefore, the lysosomal change might be a primary event in BCR‐induced apoptosis in DT40 cells. The increased activity of cathepsin B and apoptosis‐preventing effect of a cathepsin inhibitor suggested a significant role of lysosomal enzymes in this apoptosis. By microscopic studies, lysosomes of wild‐type DT40 cells fused to BCR‐carrying endosomes became enlarged and accumulated one another. In contrast, these changes of lysosomal dynamics did not occur in Syk‐deficient cells but transfer of wild‐type Syk restored the lysosomal changes and apoptosis. These results demonstrated that the lysosomal change accompanied with the activation of lysosomal enzymes is a primary step in BCR‐crosslinking‐mediated apoptosis and Syk is responsible for this step through the fusion of BCR‐carrying endosomes to lysosomes.

A novel interplay between the Fanconi anemia core complex and ATR-ATRIP kinase during DNA cross-link repair

When DNA replication is stalled at sites of DNA damage, a cascade of responses is activated in the cell to halt cell cycle progression and promote DNA repair. A pathway initiated by the kinase Ataxia teleangiectasia and Rad3 related (ATR) and its partner ATR interacting protein (ATRIP) plays an important role in this response. The Fanconi anemia (FA) pathway is also activated following genomic stress, and defects in this pathway cause a cancer-prone hematologic disorder in humans. Little is known about how these two pathways are coordinated. We report here that following cellular exposure to DNA cross-linking damage, the FA core complex enhances binding and localization of ATRIP within damaged chromatin. In cells lacking the core complex, ATR-mediated phosphorylation of two functional response targets, ATRIP and FANCI, is defective. We also provide evidence that the canonical ATR activation pathway involving RAD17 and TOPBP1 is largely dispensable for the FA pathway activation. Indeed DT40 mutant cells lacking both RAD17 and FANCD2 were synergistically more sensitive to cisplatin compared with either single mutant. Collectively, these data reveal new aspects of the interplay between regulation of ATR-ATRIP kinase and activation of the FA pathway.

c-ABL tyrosine kinase stabilizes RAD51 chromatin association.

The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR.

ATM mediates pRB function to control DNMT1 protein stability and DNA methylation.

ABSTRACT The retinoblastoma tumor suppressor gene (RB) product has been implicated in epigenetic control of gene expression owing to its ability to physically bind to many chromatin modifiers. However, the biological and clinical significance of this activity was not well elucidated. To address this, we performed genetic and epigenetic analyses in an Rb-deficient mouse thyroid C cell tumor model. Here we report that the genetic interaction of Rb and ATM regulates DNMT1 protein stability and hence controls the DNA methylation status in the promoters of at least the Ink4a, Shc2, FoxO6, and Noggin genes. Furthermore, we demonstrate that inactivation of pRB promotes Tip60 (acetyltransferase)-dependent ATM activation; allows activated ATM to physically bind to DNMT1, forming a complex with Tip60 and UHRF1 (E3 ligase); and consequently accelerates DNMT1 ubiquitination driven by Tip60-dependent acetylation. Our results indicate that inactivation of the pRB pathway in coordination with aberration in the DNA damage response deregulates DNMT1 stability, leading to an abnormal DNA methylation pattern and malignant progression.

Dual functions of ASCIZ in the DNA base damage response and pulmonary organogenesis

Zn2+-finger proteins comprise one of the largest protein superfamilies with diverse biological functions. The ATM substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN and ZNF822) was originally linked to functions in the DNA base damage response and has also been proposed to be an essential cofactor of the ATM kinase. Here we show that absence of ASCIZ leads to p53-independent late-embryonic lethality in mice. Asciz-deficient primary fibroblasts exhibit increased sensitivity to DNA base damaging agents MMS and H2O2, but Asciz deletion or knock-down does not affect ATM levels and activation in mouse, chicken, or human cells. Unexpectedly, Asciz-deficient embryos also exhibit severe respiratory tract defects with complete pulmonary agenesis and severe tracheal atresia. Nkx2.1-expressing respiratory precursors are still specified in the absence of ASCIZ, but fail to segregate properly within the ventral foregut, and as a consequence lung buds never form and separation of the trachea from the oesophagus stalls early. Comparison of phenotypes suggests that ASCIZ functions between Wnt2-2b/ß-catenin and FGF10/FGF-receptor 2b signaling pathways in the mesodermal/endodermal crosstalk regulating early respiratory development. We also find that ASCIZ can activate expression of reporter genes via its SQ/TQ-cluster domain in vitro, suggesting that it may exert its developmental functions as a transcription factor. Altogether, the data indicate that, in addition to its role in the DNA base damage response, ASCIZ has separate developmental functions as an essential regulator of respiratory organogenesis.

ATM activation by a sulfhydryl‐reactive inflammatory cyclopentenone prostaglandin

ATM (ataxia‐telangiectasia mutated) is activated by a variety of noxious agent, including oxidative stress, and ATM deficiency results in an anomalous cellular response to oxidative stress. However, the mechanisms for ATM activation by oxidative stress remain to be established. Furthermore, it is not clear whether ATM responds to oxidative DNA damage or to a change in the intracellular redox state, independent of DNA damage. We found that ATM is activated by N‐methyl‐N′‐nitro‐nitrosoguanidine (MNNG) and 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2), in NBS1‐ or MSH6‐deficient cells. We further found that ATM is activated by treating chromatin‐free immunoprecipitated ATM with MNNG or 15d‐PGJ2, which modifies free sulfhydryl (SH) groups, and that 15d‐PGJ2 binds covalently to ATM. Interestingly, 15d‐PGJ2‐induced ATM activation leads to p53 activation and apoptosis, but not to Chk2 or H2AX phosphorylation. These results indicate that ATM is activated through the direct modification of its SH groups, independent of DNA damage, and this activation leads, downstream, to apoptosis.

Rad9 modulates the P21WAF1 pathway by direct association with p53

BackgroundPrevious studies suggest that human RAD9 (hRad9), encoding a DNA damage checkpoint molecule, which is frequently amplified in epithelial tumor cells of breast, lung, head and neck cancer, participates in regulation of the tumor suppressor p53-dependent transactivation of pro-survival P21WAF1. This study examined the exact mechanism of the hRad9 function, especially through the phosphorylation of the C-terminus, in the transcription regulation of P21WAF1.ResultsThe transfection of phosphorylation-defective hRAD9 mutants of C-terminus resulted in reduction of the p53-dependent P21WAF1transactivation; the knockdown of total hRad9 elicited an increased P21WAF1mRNA expression. Immunoprecipitation and a ChIP assay showed that hRad9 and p53 formed a complex and both were associated with two p53-consensus DNA-binding sequences in the 5 region of P21WAF1gene. The association was reduced in the experiment of phosphorylation-defective hRAD9 mutants.ConclusionThe present study indicates the direct involvement of hRad9 in the p53-dependent P21WAF1transcriptional mechanism, presumably via the phosphorylation sites, and alterations of the hRad9 pathway might therefore contribute to the perturbation of checkpoint activation in cancer cells.