Hiroko Shimizu

Involvement of a NF-kappa B-like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines.

Interleukin-6 (IL-6) is one of the major mediators of inflammation, and its expression is inducible by the other inflammatory lymphokines, interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha). We demonstrate that a common IL-6 promoter element, termed inflammatory lymphokine-responsive element (ILRE), is important for induction of IL-6 gene expression by IL-1 and TNF-alpha despite possible differences in the mechanisms of action of these lymphokines. Remarkably, the ILRE sequence, located between -73 to -63 relative to the mRNA cap site, is highly homologous to NF-kappa B transcription factor-binding motifs and binds an IL-1-TNF-alpha-inducible nuclear factor; the sequence specificities, binding characteristics, and subcellular localizations of this factor are indistinguishable from those of NF-kappa B. In addition, mutations of the ILRE sequence which impair the binding of this nuclear factor abolished the induction of IL-6 gene expression by IL-1 and TNF-alpha in vivo. These results indicate that a nuclear factor indistinguishable from NF-kappa B is involved in the transcriptional activation of the IL-6 gene by IL-1 and TNF-alpha.

Disruption of ATM in p53-null cells causes multiple functional abnormalities in cellular response to ionizing radiation

ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM−/− DT40 cells displayed retarded cellular proliferation, defective G2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM−/− DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM−/− DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.

IDENTIFICATION OF A NOVEL P53 PROMOTER ELEMENT INVOLVED IN GENOTOXIC STRESS-INDUCIBLE P53 GENE EXPRESSION

p53 is recruited in response to DNA-damaging genotoxic stress and plays an important role in maintaining the integrity of the genome. We show that exposure of cells to various genotoxic agents, including anticancer drugs such as mitomycin and 5-fluorouracil, results in an increase in p53 mRNA levels and in p53 promoter activation, indicating that the p53 genotoxic stress response is partly regulated at the transcriptional level. The results of the p53 promoter analysis show that a novel p53 promoter element, termed a p53 core promoter element (from -70 to -46), is essential for basal p53 promoter activity and promoter activation induced by genotoxic agents such as anticancer drugs and UV. Although a kappa B motif partially overlaps with this element and those genotoxic agents activate NF-kappa B, it does not play a major role in p53 genotoxic stress response: NF-kappa B p65 expression did not induce significant p53 promoter activation, and NF-kappa B inhibitors (N-acetyl cysteine and I kappa B alpha) did not inhibit genotoxic stress-inducible p53 promoter activation. Finally, we characterized nuclear factors, the binding of which to the p53 core promoter element is essential for basal p53 promoter activity and p53 promoter activation induced by genotoxic agents.

A lymphoid cell-specific nuclear factor containing c-Rel-like proteins preferentially interacts with interleukin-6 kappa B-related motifs whose activities are repressed in lymphoid cells.

The proto-oncoprotein c-Rel is a member of the nuclear factor kappa B transcription factor family, which includes the p50 and p65 subunits of nuclear factor kappa B. We show here that c-Rel binds to kappa B sites as homodimers as well as heterodimers with p50. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various kappa B motifs. In particular, the c-Rel homodimer has a high affinity for interleukin-6 (IL-6) and beta interferon kappa B sites. In spite of its association with p50 in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains c-Rel but not p50 epitopes; this factor, termed IL-6 kappa B binding factor II, appears to contain the c-Rel homodimer and preferentially recognizes several IL-6 kappa B-related kappa B motifs. Although it has been previously shown that the IL-6 kappa B motif functions as a potent IL-1/tumor necrosis factor-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that IL-6 kappa B binding factor II functions as a repressor specific for IL-6 kappa B-related kappa B motifs in lymphoid cells.

53BP1 contributes to survival of cells irradiated with X-ray during G1 without Ku70 or Artemis.

Ionizing radiation (IR) induces a variety of DNA lesions. The most significant lesion is a DNA double‐strand break (DSB), which is repaired by homologous recombination or nonhomologous end joining (NHEJ) pathway. Since we previously demonstrated that IR‐responsive protein 53BP1 specifically enhances activity of DNA ligase IV, a DNA ligase required for NHEJ, we investigated responses of 53BP1‐deficient chicken DT40 cells to IR. 53BP1‐deficient cells showed increased sensitivity to X‐rays during G1 phase. Although intra‐S and G2/M checkpoints were intact, the frequency of isochromatid‐type chromosomal aberrations was elevated after irradiation in 53BP1‐deficient cells. Furthermore, the disappearance of X‐ray‐induced γ‐H2AX foci, a marker of DNA DSBs, was prolonged in 53BP1‐deficient cells. Thus, the elevated X‐ray sensitivity in G1 phase cells was attributable to repair defect for IR‐induced DNA‐damage. Epistasis analysis revealed that 53BP1 plays a role in a pathway distinct from the Ku‐dependent and Artemis‐dependent NHEJ pathways, but requires DNA ligase IV. Strikingly, disruption of the 53BP1 gene together with inhibition of phosphatidylinositol 3‐kinase family by wortmannin completely abolished colony formation by cells irradiated during G1 phase. These results demonstrate that the 53BP1‐dependent repair pathway is important for survival of cells irradiated with IR during the G1 phase of the cell cycle.

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.

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 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.

Arg tyrosine kinase is involved in homologous recombinational DNA repair.

c-Abl plays important roles in cellular response to DNA damage. However, possible roles for Arg (Abl-related gene) in DNA damage response are unknown. Here, we show that ionizing radiation (IR)-induced Rad51 focus formation is reduced in Arg-deficient cells generated from a chicken B cell line by targeted disruption. This is consistent with the findings that Arg-deficient cells display hypersensitivity to IR, elevated frequencies of IR-induced chromosomal aberrations, and reduced targeted integration frequencies. All of these abnormalities in DNA damage repair are also observed in ATM-deficient cells but not in c-Abl-deficient cells. Finally, we show that Arg interacts with and phosphorylates Rad51 in 293T cells. These results suggest that Arg plays a role in homologous recombinational (HR) DNA repair by phosphorylating Rad51.

Detection of c-Abl kinase-promoted phosphorylation of Rad51 by specific antibodies reveals that Y54 phosphorylation is dependent on that of Y315

MINT‐7034009: cABL (uniprotkb:P00519) physically interacts (MI:0218) with RAD51 (uniprotkb:Q06609) by pull down (MI:0096)