Eiko Takada

Prevention of Anti-IgM-Induced Apoptosis Accompanying G1 Arrest in B Lymphoma Cells Overexpressing Dominant-Negative Mutant Form of c-Jun N-Terminal Kinase 1

A family of mitogen-activated protein (MAP) kinases comprising the extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 MAP kinases are involved in proliferation and apoptosis. However, there are some arguments concerning the role of these kinases in Ag-induced B cell apoptosis. Two of the B lymphoma cell lines (CH31 and WEHI-231) susceptible to anti-IgM-induced apoptosis were used as a model. To address these issues, we examined the kinetics of anti-IgM-induced activation of MAP kinases and established cell lines overexpressing a dominant-negative (dn) mutant form of JNK1 (dnJNK1). Anti-IgM induced a sustained JNK1 activation with a peak at 8 h, with a marginal activation of ERK1/ERK2 in CH31 cells. The sustained JNK1 activation was not a secondary event through a caspase activation. The peak point of the JNK1 activation was just before the onset of a decline in mitochondrial membrane potential, which preceded anti-IgM-induced cell death. Following anti-IgM stimulation, dnJNK1 prevented a decline in mitochondrial membrane potential at 24 h, with a prolonged inhibition up to 72 h in WEHI-231, although it did so only partially during a later time period in CH31. The dnJNK1 cells also demonstrated diminished procaspase-3 activation and a decreased rate of apoptosis upon anti-IgM stimulation, with a concomitant increased arrest in G1 phase, which could be explained by enhanced levels of cyclin-dependent kinase inhibitor p27Kip1 protein. Thus, anti-IgM-induced JNK activation might be implicated in cell cycle progression as well as in apoptosis regulation, probably involving p27Kip1 protein.

Chemotherapeutic agents sensitize sarcoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand-induced caspase-8 activation, apoptosis and loss of mitochondrial membrane potential

Chemotherapeutic agents have been used for the treatment of patients with osteosarcoma (OS). However, inherent or acquired resistance to these agents is a serious problem in the management of OS patients. Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is considered to induce apoptosis in a variety of cancer cells but not normal cells. In the present study, we examined whether chemotherapeutic agents enhance TRAIL‐induced apoptosis in the sarcoma cell lines MG‐63 and SaOS‐2. Pretreatment with sub‐toxic or slightly toxic concentrations of chemotherapeutic agents (cis‐diammine dichloroplatinum, CDDP and doxorubicin, DXR) sensitized both cell lines to TRAIL‐induced apoptosis, as assessed by the propidium iodide or Annexin V‐Cy5 staining method. These cell lines expressed death receptors TRAIL‐receptor 1 (TRAIL‐R1) and TRAIL‐R2, which were unaltered by treatment with CDDP, as assessed by flow cytometry. The decoy receptors TRAIL‐R3 and ‐R4 were barely detected in both cell lines. CDDP down‐regulated c‐FLIP, tending to lower the activation threshold required for TRAIL‐induced caspase‐8 activation. The CDDP‐pretreated cells indeed demonstrated more increased TRAIL‐mediated caspase‐8 activation, loss of mitochondrial membrane potential (ΔΨm), and apoptosis than untreated cells. Consequently, the activated caspase‐8 might lead to either activation of effector caspases such as caspase‐3 or loss in ΔΨm. Both the increased caspase activation and mitochondrial dysfunction induced by combination of CDDP and TRAIL would contribute to enhanced apoptotic cell death. The results of the present study would be valuable for the design of novel treatment modalities for patients with OS.

Environment-induced epigenetic reprogramming in genomic regulatory elements in smoking mothers and their children

Epigenetic mechanisms have emerged as links between prenatal environmental exposure and disease risk later in life. Here, we studied epigenetic changes associated with maternal smoking at base pair resolution by mapping DNA methylation, histone modifications, and transcription in expectant mothers and their newborn children. We found extensive global differential methylation and carefully evaluated these changes to separate environment associated from genotype‐related DNA methylation changes. Differential methylation is enriched in enhancer elements and targets in particular “commuting” enhancers having multiple, regulatory interactions with distal genes. Longitudinal whole‐genome bisulfite sequencing revealed that DNA methylation changes associated with maternal smoking persist over years of life. Particularly in children prenatal environmental exposure leads to chromatin transitions into a hyperactive state. Combined DNA methylation, histone modification, and gene expression analyses indicate that differential methylation in enhancer regions is more often functionally translated than methylation changes in promoters or non‐regulatory elements. Finally, we show that epigenetic deregulation of a commuting enhancer targeting c‐Jun N‐terminal kinase 2 (JNK2) is linked to impaired lung function in early childhood.

Cleavage of Bax-α and Bcl-xL during carboplatin-mediated apoptosis in squamous cell carcinoma cell line

Abstract Although carboplatin (CBDCA) has been used for the treatment of several types of tumors, the complete response rate has been limited, probably because of inherent or CBDCA-induced resistance. As a first step to overcome these problems, we tried to elucidate the mechanisms of CBDCA-mediated cytotoxicity in the squamous cell carcinoma cell line MIT7. The treatment of cells with CBDCA resulted in apoptosis in a dose-dependent manner, as assessed by the propidium iodide staining method and DNA degradation in a nucleosomal pattern. The induction of apoptosis was accompanied by the decline of mitochondrial membrane potential (Δψm ) at 12 h following CBDCA stimulation. Variant forms of p18 Bax-α and p16 Bcl-xL were generated with the down-regulation of both Bax-α (p21) and Bcl-xL (p31) at 36 and 48 h following CBDCA stimulation, suggesting that the modulation of Bcl-2 family proteins Bax-α and Bcl-xL play some role in CBDCA-mediated apoptosis. The activation of caspase-3 and -8 occurred at 12 and 24 h following the stimulation, respectively. The pretreatment of cells with pan-caspase inhibitor Z-VAD-fmk markedly prevented CBDCA-mediated cytotoxicity/apoptosis and the modulation of Bcl-2 family proteins (generation of p18 Bax-α and p16 Bcl-xL ) with only slight prevention of decline of Δψm. Taken together, these results may suggest that activation of several caspases, including caspase-3 and -8, plays some role in CBDCA-mediated apoptosis, probably through the modification of Bcl-2 family proteins, Bax-α and Bcl-xL. Moreover, caspase activation may occur downstream of membrane depolarization.

Apoptosis signal-regulating kinase (ASK)-1 mediates apoptosis through activation of JNK1 following engagement of membrane immunoglobulin.

Engagement of membrane immunoglobulin (mIg) on WEHI-231 mouse B lymphoma cells results in growth arrest at the G1 phase of the cell cycle, followed by a reduction of mitochondrial membrane potential (DeltaPsim) and apoptosis. WEHI-231 cells resemble immature B cells in terms of the cell surface phenotype and sensitivity to mIg engagement. However, the molecular mechanisms underlying mIg-induced loss of DeltaPsim and apoptosis have not yet been established. In this study, we show that apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase 1 (JNK1) signaling pathway participates in mIg-induced apoptosis through the generation of reactive oxygen species (ROS). Stimulation of WEHI-231 cells with anti-IgM induces phosphorylation and subsequent activation of ASK1, leading to JNK activation. Anti-IgM stimulation immediately (5 min) induces hydrogen peroxide (H2O2) production with a substantial increase during later time points (36-48 h), accompanied by loss of DeltaPsim and an increase in cells with sub-G1 DNA content. The anti-IgM-induced late-phase H2O2 production, loss of DeltaPsim, and increase in the sub-G1 fraction were all reduced substantially in WEHI-231 cells overexpressing a dominant-negative form of ASK1, compared with control vector alone, but enhanced substantially in cells overexpressing a constitutively active form of ASK1. These mIg-mediated events were also partially abrogated by ROS scavenger N-acetyl-L-cysteine (NAC). Taken together, these results suggest that mIg engagement induces H2O2 production leading to activation of ASK1-JNK1 pathway, creating a feedback amplification loop of ROS-ASK/JNK that leads to loss of DeltaPsim and finally apoptosis.

Blockade of the OX40 ligand prolongs corneal allograft survival

Although corneal transplantation is one of the most common tissue transplantations and is known to have a high graft acceptance rate, occasional corneal graft rejection remains a cause of blindness. OX40, a member of the TNF receptor superfamily, is expressed on activated T cells, and transmits a costimulatory signal by binding to OX40 ligand (OX40L) expressed on several cells with antigen‐presenting functions. Using a blocking monoclonal antibody (mAb) against murine OX40L, we investigated the role of OX40 in a murine model of corneal transplantation. C3H/He mouse corneas were transplanted to BALB/c mice orthotopically. Administration of anti‐OX40L mAb significantly reduced allograft rejection, and increased graft survival rate to 40% at 8 weeks after transplantation, while all corneas were rejected within 5 weeks in control IgG‐treated mice. Similar reduced rejection was observed when wild‐type donor corneas were transplanted to OX40L‐deficient recipients. In vitro study revealed that the anti‐OX40L mAb treatment reduced proliferative response and IFN‐γ production of draining lymph node cells in response to stimulation with donor alloantigen. These results demonstrate that OX40L blockade is effective for prolongation of corneal allograft survival by inhibiting recipient T cell activation.

c-Jun-NH2-terminal kinase potentiates apoptotic cell death in response to carboplatin in B lymphoma cells

PurposeExposure to carboplatin (CBDCA) has been demonstrated to result in apoptotic and/or necrotic cell death, but molecular mechanisms underlying CBDCA-induced apoptosis or necrosis remain largely unclear. Here, we examined whether activation of c-Jun NH2-terminal kinase (JNK) modulates the mode of cell death induced by CBDCA in CD31 B lymphoma cells.MethodsThe mode of cell death (apoptosis versus necrosis) was investigated by flow cytometry using 7-amino-actinomycin D (7-AAD) and annexin-FITC probes. To evaluate the role of JNK1 in CBDCA-induced cell death, CH31 B lymphoma cells overexpressing dominant-negative form of JNK1 (dnJNK1) or constitutively active form of JNK1 (MKK7-JNK1) were established. Intracellular accumulation of superoxide anion (O2−) was determined by flow cytometry using the fluorescent probe dihydroethidium (DHE).ResultsThe CBDCA-induced primary apoptosis and secondary necrosis were abrogated in the dnJNK1-overexpressing CH31 cells, while it was somewhat enhanced in the MKK7-JNK1-overexpressing cells. In contrast, the CBDCA-induced primary necrosis was reduced by MKK7-JNK1, with a concurrent decrease in production of O2−. The superoxide anion scavenger for butylated hydroxyanisol (BHA) partially reduced the CBDCA-induced O2− production and necrotic, but not apoptotic, death in both wild type and dnJNK1-overexpressing CH31 cells.ConclusionsProlonged activation of JNK1 appears to be involved in CBDCA-induced apoptosis with prevention of necrosis induction, and the induction of necrosis appears to correlate with CBDCA-induced O2− production, which is partially blocked by co-culture with BHA. These observations provide valuable information for understanding molecular mechanisms underlying CBDCA-induced cell death, and hopefully for the design of novel treatment modalities for patients with tumors.

Requirement of Apoptosis-Inducing Kinase 1 for the Induction of Bronchial Asthma following Stimulation with Ovalbumin

Background: Bronchial asthma is a chronic inflammatory disease of the airway. Apoptosis signal-regulating kinase 1 (ASK1), a member of the mitogen-activated protein kinase kinase kinase family, is activated by environmental stress and plays a crucial role in the induction of apoptosis and inflammation. To examine whether ASK1 is involved in the induction of bronchial asthma, we investigated the role of ASK1 using a genetic approach in the production of cytokines, as well as the development of airway hyperreactivity (AHR) and antibody responses using a murine airway inflammation model. Methods: ASK1-deficient (ASK1-/-) and control wild-type (WT) mice were immunized with ovalbumin (OVA) without alum intraperitoneally, followed by intranasal administration of OVA. Airway infiltration of inflammatory cells, cytokine production, AHR and antibody production were assayed. The asthmatic phenotype was assessed following intranasal administration of IL-13 or TNF-α. Results: ASK1-/- mice sensitized with OVA displayed an impaired inflammatory cell infiltration into airways and a decreased AHR relative to WT mice. Moreover, the production of OVA-specific IgE antibodies and proasthmatic cytokines (IL-5, IL-13 and TNF-α) was substantially reduced in OVA-stimulated ASK1-/- mice. Intranasal administration of IL-13 and OVA enhanced the accumulation of inflammatory cells in OVA-primed ASK1-/- mice. The OVA-induced AHR in response to methacholine was enhanced by IL-13 in WT mice but not ASK1-/- mice. Conclusions: The ASK1 signaling pathway regulates the OVA-induced asthmatic phenotype, specifically AHR sensitivity and cytokine production. Therefore, the ASK1 signaling pathway is a promising target for therapeutic intervention in some asthmatic patients.

c‐Jun NH2‐terminal kinase (JNK)‐dependent nuclear translocation of apoptosis‐inducing factor (AIF) following engagement of membrane immunoglobulin on WEHI‐231 B lymphoma cells

WEHI‐231 B lymphoma cells have been employed for analysis of antigen‐induced B cell unresponsiveness because these cells undergo cell cycle arrest in G1, accompanied by induction of apoptosis. In the present study, we examined the requirement for toxic small molecules apoptosis‐inducing factor (AIF) and cytochrome c, and subsequent caspase activation in apoptotic cell death in WEHI‐231 and CH31 B lymphoma cells following engagement of membrane immunoglobulin (mIg). Pan‐caspase inhibitor BD‐fmk blocked mIg‐mediated increase in cells with sub‐G1 DNA content, whereas it did not affect mIg‐mediated loss of mitochondrial membrane potential and phosphatidylserine exposure on B cell membrane. Dominant‐negative form of c‐Jun NH2‐terminal kinase1 (JNK1) blocked the translocation of AIF into the nuclei and cytosol from the mitochondria in the WEHI‐231 and CH31 cells following mIg engagement, whereas constitutively active form of JNK1 enhanced it. This AIF translocation was also blocked by Bcl‐xL, but not by BD‐fmk. Moreover, AIF‐deficient clones via small interfering RNA (siRNA)‐mediated method showed small increase in loss of mitochondrial membrane potential. After mIg engagement, the AIF‐deficient clones displayed an enhanced sensitivity to mIg‐mediated apoptosis, concomitant with translocation of a residual AIF into the nuclei, compared with control clone. Our findings are compatible with the notion that AIF has dual role, with a proapoptotic function in the nuclei and a distinct anti‐apoptotic function in the mitochondria. These observations would be valuable for analysis of B cell unresponsiveness and hopefully for treatment of diseases involving B cell dysfunction. J. Cell. Biochem. 104: 1927–1936, 2008.

Oral administration of interferon-β suppresses experimental autoimmune uveoretinitis

Abstract.Background: The oral administration of type I interferons (IFNs) have been reported to reduce severity of inflammation in several animal models of autoimmune disease. This study examined whether oral administration of IFN-β is capable of modulating inflammation in experimental autoimmune uveoretinitis (EAU). Methods: EAU was induced in rats by immunization with interphotoreceptor retinoid-binding protein (IRBP) emulsified in complete Freunds adjuvant. Rats were treated with either varying doses (102, 103, 104 or 105 IU) of mouse recombinant IFN-β or phosphate-buffered saline for control, via direct oropharyngeal application once a day for 28 days starting 7 days before IRBP immunization. Intraocular inflammation was assessed by slit-lamp biomicroscopy and histopathological examination. Spleen cell proliferation response and cytokine production under IRBP stimulation were assessed. Spleen cell subpopulations were also measured. Results: IFN-β at doses of either 104 or 105 IU significantly reduced both the clinical and histopathological severity of EAU. Spleen cell proliferation and IFN-γ production from rats treated with 104 IU IFN-β were significantly decreased compared with controls. Furthermore, the proportion of both NK cells and NKT cells in the spleen of rats treated with IFN-β was increased compared with controls. Conclusion: These results suggest that the oral administration of IFN-β reduces inflammation in IRBP-mediated EAU and that the mechanism of this action may involve NK cells and NKT cells.