Apoptosis Induced by Cytoskeletal Disruption Requires Distinct Domains of MEKK1

MEKK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the MAPK JNK and is required for microtubule inhibitor-induced apoptosis in B cells. Here, we find that apoptosis induced by actin disruption via cytochalasin D and by the protein phosphatase 1/2A inhibitor okadaic acid also requires MEKK1 activation. To elucidate the functional requirements for activation of the MEKK1-dependent apoptotic pathway, we created mutations within MEKK1. MEKK1-deficient cells were complemented with MEKK1 containing mutations in either the ubiquitin interacting motif (UIM), plant homeodomain (PHD), caspase cleavage site or the kinase domain at near endogenous levels of expression and tested for their sensitivity to each drug. We found that both the kinase activity and the PHD domain of MEKK1 are required for JNK activation and efficient induction of apoptosis by drugs causing cytoskeletal disruption. Furthermore, we discovered that modification of MEKK1 and its localization depends on the integrity of the PHD

Prognostic factors influencing clinical outcome of allogeneic hematopoietic stem cell transplantation following imatinib-based therapy in BCR–ABL-positive ALL

We investigated prognostic factors for the clinical outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ALL) following imatinib-based therapy. Among 100 adult patients who were prospectively enrolled in the JALSG Ph+ALL202 study, 97 patients obtained complete remission (CR) by imatinib-combined chemotherapy, among whom 60 underwent allo-HSCT in their first CR. The probabilities of overall survival (OS) and disease-free survival (DFS) at 3 years after HSCT were 64% (95% CI, 49–76) and 58% (95% CI, 43–70), respectively. Prognostic factor analysis revealed that the major BCR–ABL transcript was the only unfavorable predictor for OS and DFS after HSCT by both univariate (HR, 3.67 (95% CI 1.49–9.08); P=0.005 and HR, 6.25 (95% CI, 1.88–20.8); P=0.003, respectively) and multivariate analyses (HR, 3.20 (95% CI, 1.21–8.50); P=0.019 and HR, 6.92 (95% CI, 2.09–22.9); P=0.002, respectively). Minimal residual disease status at the time of HSCT had a significant influence on relapse rate (P=0.015). Further study of the BCR–ABL subtype for the clinical impact on outcome of allo-HSCT in Ph+ALL is warranted

Minor Abnormalities of Testis Development in Mice Lacking the Gene Encoding the MAPK Signalling Component, MAP3K1

In mammals, the Y chromosome is a dominant male determinant, causing the bipotential gonad to develop as a testis. Recently, cases of familial and spontaneous 46,XY disorders of sex development (DSD) have been attributed to mutations in the human gene encoding mitogen-activated protein kinase kinase kinase 1, MAP3K1, a component of the mitogen-activated protein kinase (MAPK) signal transduction pathway. In individuals harbouring heterozygous mutations in MAP3K1, dysregulation of MAPK signalling was observed in lymphoblastoid cell lines, suggesting a causal role for these mutations in disrupting XY sexual development. Mice lacking the cognate gene, Map3k1, are viable and exhibit the eyes open at birth (EOB) phenotype on a mixed genetic background, but on the C57BL/6J genetic background most mice die at around 14.5 dpc due to a failure of erythropoiesis in the fetal liver. However, no systematic examination of sexual development in Map3k1-deficient mice has been described, an omission that is especially relevant in the case of C57BL/6J, a genetic background that is sensitized to disruptions to testis determination. Here, we report that on a mixed genetic background mice lacking Map3k1 are fertile and exhibit no overt abnormalities of testis development. On C57BL/6J, significant non-viability is observed with very few animals surviving to adulthood. However, an examination of development in Map3k1-deficient XY embryos on this genetic background revealed no significant defects in testis determination, although minor abnormalities were observed, including an increase in gonadal length. Based on these observations, we conclude that MAP3K1 is not required for mouse testis determination. We discuss the significance of these data for the functional interpretation of sex-reversing MAP3K1 mutations in humans

TRAF6 Establishes Innate Immune Responses by Activating NF-κB and IRF7 upon Sensing Cytosolic Viral RNA and DNA

BACKGROUND:In response to viral infection, the innate immune system recognizes viral nucleic acids and then induces production of proinflammatory cytokines and type I interferons (IFNs). Toll-like receptor 7 (TLR7) and TLR9 detect viral RNA and DNA, respectively, in endosomal compartments, leading to the activation of nuclear factor kappaB (NF-kappaB) and IFN regulatory factors (IRFs) in plasmacytoid dendritic cells. During such TLR signaling, TNF receptor-associated factor 6 (TRAF6) is essential for the activation of NF-kappaB and the production of type I IFN. In contrast, RIG-like helicases (RLHs), cytosolic RNA sensors, are indispensable for antiviral responses in conventional dendritic cells, macrophages, and fibroblasts. However, the contribution of TRAF6 to the detection of cytosolic viral nucleic acids has been controversial, and the involvement of TRAF6 in IRF activation has not been adequately addressed. PRINCIPAL FINDINGS:Here we first show that TRAF6 plays a critical role in RLH signaling. The absence of TRAF6 resulted in enhanced viral replication and a significant reduction in the production of IL-6 and type I IFNs after infection with RNA virus. Activation of NF-kappaB and IRF7, but not that of IRF3, was significantly impaired during RLH signaling in the absence of TRAF6. TGFbeta-activated kinase 1 (TAK1) and MEKK3, whose activation by TRAF6 during TLR signaling is involved in NF-kappaB activation, were not essential for RLH-mediated NF-kappaB activation. We also demonstrate that TRAF6-deficiency impaired cytosolic DNA-induced antiviral responses, and this impairment was due to defective activation of NF-kappaB and IRF7. CONCLUSIONS/SIGNIFICANCE:Thus, TRAF6 mediates antiviral responses triggered by cytosolic viral DNA and RNA in a way that differs from that associated with TLR signaling. Given its essential role in signaling by various receptors involved in the acquired immune system, TRAF6 represents a key molecule in innate and antigen-specific immune responses against viral infection

Modulating sensitivity to drug-induced apoptosis: the future for chemotherapy?

Drug resistance is a fundamental problem in the treatment of most common human cancers. Our understanding of the cellular mechanisms underlying death and survival has allowed the development of rational approaches to overcoming drug resistance. The mitogen activated protein kinase family of protein serine/threonine kinases has been implicated in this complex web of signalling, with some members acting to enhance death and other members to prevent it. A recent publication by MacKeigan et al is the first to demonstrate an enhancement of drug-induced cell death by simultaneous blockade of MEK-mediated survival signalling, and offers the potential for targeted adjuvant therapy as a means of overcoming drug resistance

The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase

<p>Abstract</p> <p>Background</p> <p>Dietary isothiocyanates (ITCs) are electrophilic compounds that have diverse biological activities including induction of apoptosis and effects on cell cycle. They protect against experimental carcinogenesis in animals, an activity believed to result from the transcriptional induction of "Phase 2" enzymes. The molecular mechanism of action of ITCs is unknown. Since ITCs are electrophiles capable of reacting with sulfhydryl groups on amino acids, we hypothesized that ITCs induce their biological effects through covalent modification of proteins, leading to changes in cell regulatory events. We previously demonstrated that stress-signaling kinase pathways are inhibited by other electrophilic compounds such as menadione. We therefore tested the effects of nutritional ITCs on MEKK1, an upstream regulator of the SAPK/JNK signal transduction pathway.</p> <p>Methods</p> <p>The activity of MEKK1 expressed in cells was monitored using in vitro kinase assays to measure changes in catalytic activity. The activity of endogenous MEKK1, immunopurified from ITC treated and untreated LnCAP cells was also measured by in vitro kinase assay. A novel labeling and affinity reagent for detection of protein modification by ITCs was synthesized and used in competition assays to monitor direct modification of MEKK1 by ITC. Finally, immunoblots with phospho-specific antibodies were used to measure the activity of MAPK protein kinases.</p> <p>Results</p> <p>ITCs inhibited the MEKK1 protein kinase in a manner dependent on a specific cysteine residue in the ATP binding pocket. Inhibition of MEKK1 catalytic activity was due to direct, covalent and irreversible modification of the MEKK1 protein itself. In addition, ITCs inhibited the catalytic activity of endogenous MEKK1. This correlated with inhibition of the downstream target of MEKK1 activity, i.e. the SAPK/JNK kinase. This inhibition was specific to SAPK, as parallel MAPK pathways were unaffected.</p> <p>Conclusion</p> <p>These results demonstrate that MEKK1 is directly modified and inhibited by ITCs, and that this correlates with inhibition of downstream activation of SAPK. These results support the conclusion that ITCs may carry out many of their actions by directly targeting important cell regulatory proteins.</p

Activation of JNK Triggers Release of Brd4 from Mitotic Chromosomes and Mediates Protection from Drug-Induced Mitotic Stress

Some anti-cancer drugs, including those that alter microtubule dynamics target mitotic cells and induce apoptosis in some cell types. However, such drugs elicit protective responses in other cell types allowing cells to escape from drug-induced mitotic inhibition. Cells with a faulty protective mechanism undergo defective mitosis, leading to genome instability. Brd4 is a double bromodomain protein that remains on chromosomes during mitosis. However, Brd4 is released from mitotic chromosomes when cells are exposed to anti-mitotic drugs including nocodazole. Neither the mechanisms, nor the biological significance of drug-induced Brd4 release has been fully understood. We found that deletion of the internal C-terminal region abolished nocodazole induced Brd4 release from mouse P19 cells. Furthermore, cells expressing truncated Brd4, unable to dissociate from chromosomes were blocked from mitotic progression and failed to complete cell division. We also found that pharmacological and peptide inhibitors of the c-jun-N-terminal kinases (JNK) pathway, but not inhibitors of other MAP kinases, prevented release of Brd4 from chromosomes. The JNK inhibitor that blocked Brd4 release also blocked mitotic progression. Further supporting the role of JNK in Brd4 release, JNK2–/– embryonic fibroblasts were defective in Brd4 release and sustained greater inhibition of cell growth after nocodazole treatment. In sum, activation of JNK pathway triggers release of Brd4 from chromosomes upon nocodazole treatment, which mediates a protective response designed to minimize drug-induced mitotic stress

Large-Scale Evidence for Conservation of NMD Candidature Across Mammals

BACKGROUND: Alternatively-spliced (AS) forms can vary protein function, intracellular localization and post-translational modifications. AS coupled with mRNA nonsense-mediated decay (NMD) can also control the transcript abundance. Here, we have investigated the genome-scale conservation of alternatively-spliced NMD candidates (AS-NMD candidates), in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We mapped>12 million cDNA/EST library transcripts, comprising pooled data from both older and next-generation sequencing techniques, against genomic sequences to annotate AS-NMD candidates generated by in-frame premature termination codons (PTCs), in the human, mouse, rat and cow genomes. In these genomes, we found populations of genes that harbour AS-NMD candidates, varying in number from approximately 149 to 2,051 genes. We discovered that a highly-significant proportion (27%-35%) of AS-NMD candidate genes in mouse, rat and cow, also have human orthologs targeted for NMD. Intron retention was the most abundant type of AS-NMD, ranging from 43% to 67% of genes harbouring an AS-NMD candidate. Groupings of AS-NMD candidate genes either with or without intron retentions also have highly significant AS-NMD conservation, indicating that the trend is not due primarily to conservation of intron retentions. As a subset, the AS-NMD intron retentions are distinguished from non-retained introns by higher GC content, and codon usage similar to the usage in protein-coding sequences. This indicates that most of these alternatively spliced sequences have coded for proteins in the recent evolutionary past. In general, the AS-NMD candidate genes showed a similar pattern of Gene Ontology functional category enrichments in all four species. Genes linked to nucleic-acid interaction and apoptosis, and involved in pathways linked with cancer, were the most common. Finally, we mapped the AS-NMD candidates to mass spectrometry-derived proteomics data, and gathered evidence of truncated polypeptides for at least 10% of all human AS-NMD candidate transcripts. CONCLUSIONS/SIGNIFICANCE: In summary, our analysis provides strong statistical evidence for conservation of functional AS-NMD candidature across Mammalia for a large subset of genes. However, because codon usage of AS-NMD intron retentions is similar to the usage in exons, it is difficult to de-couple conservation of AS-NMD-based regulation from conservation for protein-coding ability, for intron retentions

Markedly improved outcomes and acceptable toxicity in adolescents and young adults with acute lymphoblastic leukemia following treatment with a pediatric protocol: a phase II study by the Japan Adult Leukemia Study Group

The superiority of the pediatric protocol for adolescents with acute lymphoblastic leukemia (ALL) has already been demonstrated, however, its efficacy in young adults remains unclear. The ALL202-U protocol was conducted to examine the efficacy and feasibility of a pediatric protocol in adolescents and young adults (AYAs) with BCR\u27ABL-negative ALL. Patients aged 15\u2724 years (n = 139) were treated with the same protocol used for pediatric B-ALL. The primary objective of this study was to assess the disease-free survival (DFS) rate and its secondary aims were to assess toxicity, the complete remission (CR) rate and the overall survival (OS) rate. The CR rate was 94%. The 5-year DFS and OS rates were 67% (95% confidence interval (CI) 58\u2775%) and 73% (95% CI 64\u2780%), respectively. Severe adverse events were observed at a frequency that was similar to or lower than that in children treated with the same protocol. Only insufficient maintenance therapy significantly worsened the DFS (hazard ratio 5.60, Po0.001).These results indicate that this protocol may be a feasible and highly effective treatment for AYA with BCR\u27ABL-negative ALL

Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function