Cell adhesion tunes inflammatory TPL2 kinase signal transduction.

Signaling through adhesion-related molecules is important for cancer growth and metastasis and cancer cells are resistant to anoikis, a form of cell death ensued by cell detachment from the extracellular matrix. Herein, we report that detached carcinoma cells and immortalized fibroblasts display defects in TNF and CD40 ligand (CD40L)-induced MEK-ERK signaling. Cell detachment results in reduced basal levels of the MEK kinase TPL2, compromises TPL2 activation and sensitizes carcinoma cells to death-inducing receptor ligands, mimicking the synthetic lethal interactions between TPL2 inactivation and TNF or CD40L stimulation. Focal Adhesion Kinase (FAK), which is activated in focal adhesions and mediates anchorage-dependent survival signaling, was found to sustain steady state TPL2 protein levels and to be required for TNF-induced TPL2 signal transduction. We show that when FAK levels are reduced, as seen in certain types of malignancy or malignant cell populations, the formation of cIAP2:RIPK1 complexes increases, leading to reduced TPL2 expression levels by a dual mechanism: first, by the reduction in the levels of NF-κΒ1 which is required for TPL2 stability; second, by the engagement of an RelA NF-κΒ pathway that elevates interleukin-6 production, leading to activation of STAT3 and its transcriptional target SKP2 which functions as a TPL2 E3 ubiquitin ligase. These data underscore a new mode of regulation of TNF family signal transduction on the TPL2-MEK-ERK branch by adhesion-related molecules that may have important ramifications for cancer therapy.

NSP9 of SARS-CoV-2 attenuates nuclear transport by hampering nucleoporin 62 dynamics and functions in host cells.

Nuclear pore complexes (NPC) regulate molecular traffics on nuclear envelope, which plays crucial roles during cell fate specification and diseases. The viral accessory protein NSP9 of SARS-CoV-2 is reported to interact with nucleoporin 62 (NUP62), a structural component of the NPC, but its biological impact on the host cell remain obscure. Here, we established new cell line models with ectopic NSP9 expression and determined the subcellular destination and biological functions of NSP9. Confocal imaging identified NSP9 to be largely localized in close proximity to the endoplasmic reticulum. In agreement with the subcellular distribution of NSP9, association of NSP9 with NUP62 was observed in cytoplasm. Furthermore, the overexpression of NSP9 correlated with a reduction of NUP62 expression on the nuclear envelope, suggesting that attenuating NUP62 expression might have contributed to defective NPC formation. Importantly, the loss of NUP62 impaired translocation of p65, a subunit of NF-κB, upon TNF-α stimulation. Concordantly, NSP9 over-expression blocked p65 nuclear transport. Taken together, these data shed light on the molecular mechanisms underlying the modulation of host cells during SARS-CoV-2 infection.

The potential role of chitosan-based nanoparticles as drug delivery systems in pancreatic cancer.

Pancreatic cancer (PC) is one of the most lethal cancers and 12th most common cancer in the world. Due to the inaccessible anatomical position of the pancreas and asymptomatic early stages of this disease, PC has a high mortality rate. Therefore, providing reliable diagnostic and therapeutic tools are the keys to increase the PC survival rate. Nanotechnology is an inchoate field of science that previously scientists' tendency to enhance the efficacy of current preventive, diagnostic, and therapeutic methods has oriented them to build a bridge between this science and medicine. In the case of PC, nanotechnology suggests using drug delivery devices for a more effective and targeted therapy. Chitosan is a natural polymer that recently has attracted a lot of attention for being renewable, nontoxic, and bioabsorbable. In this article, we tend to look for the answer to this question: has nanotechnology been successful in using chitosan-based nanoformulations as carriers for preventing more individuals from suffering or at least increasing the 5-year survival of the PC patients?

MicroRNA-577 promotes the sensitivity of chronic myeloid leukemia cells to imatinib by targeting NUP160.

OBJECTIVE: To explore the effect of microRNA-577 on the drug sensitivity of chronic myeloid leukemia (CML) and the underlying mechanism. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression of microRNA-577 in peripheral blood of patients with chronic myeloid leukemia. Meanwhile, the expression of microRNA-577 was detected in CML cell line after imatinib treatment. Cell counting kit-8 (CCK-8) and flow cytometry assay were applied to verify the effect of microRNA-577 on cell proliferation and cycle. NUP160 was identified as a target gene of microRNA-577 by dual-luciferase reporter gene assay. Cell reverse test was performed to figure out whether microRNA-577 can enhance the sensitivity of CML to imatinib. RESULTS: QRT-PCR results revealed that microRNA-577 level was notably decreased in peripheral blood of patients with CML, and microRNA-577 could inhibit the proliferation and cycle of CML cells. In addition, the result of dual-luciferase reporting assay indicated that microRNA-577 had a binding relationship with NUP160, and up-regulation of microRNA-577 in CML cell lines reduced the expression of NUP160, and vice versa. Lastly, cell reverse experiments confirmed that microRNA-577 can alleviate the resistance of CML to imatinib. CONCLUSIONS: We found that microRNA-577 promotes the sensitivity of chronic myeloid leukemia cells to imatinib by down-regulating the expression of NUP160.

The nuclear pore proteins Nup88/214 and T-cell acute lymphatic leukemia-associated NUP214 fusion proteins regulate Notch signaling.

The Notch receptor is a key mediator of developmental programs and cell-fate decisions. Imbalanced Notch signaling leads to developmental disorders and cancer. To fully characterize the Notch signaling pathway and exploit it in novel therapeutic interventions, a comprehensive view on the regulation and requirements of Notch signaling is needed. Notch is regulated at different levels, ranging from ligand binding, stability to endocytosis. Using an array of different techniques, including reporter gene assays, immunocytochemistry, and ChIP-qPCR we show here, to the best of our knowledge for the first time, regulation of Notch signaling at the level of the nuclear pore. We found that the nuclear pore protein Nup214 (nucleoporin 214) and its interaction partner Nup88 negatively regulate Notch signaling in vitro and in vivo in zebrafish. In mammalian cells, loss of Nup88/214 inhibited nuclear export of recombination signal-binding protein for immunoglobulin κJ region (RBP-J), the DNA-binding component of the Notch pathway. This inhibition increased binding of RBP-J to its cognate promoter regions, resulting in increased downstream Notch signaling. Interestingly, we also found that NUP214 fusion proteins, causative for certain cases of T-cell acute lymphatic leukemia, potentially contribute to tumorigenesis via a Notch-dependent mechanism. In summary, the nuclear pore components Nup88/214 suppress Notch signaling in vitro, and in zebrafish, nuclear RBP-J levels are rate-limiting factors for Notch signaling in mammalian cells, and regulation of nucleocytoplasmic transport of RBP-J may contribute to fine-tuning Notch activity in cells.

RIP1/RIP3-regulated necroptosis as a target for multifaceted disease therapy (Review).

Necroptosis is a type of programmed cell death with necrotic morphology, occurring in a variety of biological processes, including inflammation, immune response, embryonic development and metabolic abnormalities. The current nomenclature defines necroptosis as cell death mediated by signal transduction from receptor­interacting serine/threonine kinase (RIP) 1 to RIP3 (hereafter called RIP1/RIP3). However, RIP3­dependent cell death would be a more precise definition of necroptosis. RIP3 is indispensable for necroptosis, while RIP1 is not consistently involved in the signal transduction. Notably, deletion of RIP1 even promotes RIP3­mediated necroptosis under certain conditions. Necroptosis was previously thought as an alternate process of cell death in case of apoptosis inhibition. Currently, necroptosis is recognized to serve a pivotal role in regulating various physiological processes. Of note, it mediates a variety of human diseases, such as ischemic brain injury, immune system disorders and cancer. Targeting and inhibiting necroptosis, therefore, has the potential to be used for therapeutic purposes. To date, research has elucidated the suppression of RIP1/RIP3 via effective inhibitors and highlighted their potential application in disease therapy. The present review focused on the molecular mechanisms of RIP1/RIP3­mediated necroptosis, explored the functions of RIP1/RIP3 in necroptosis, discussed their potential as a novel therapeutic target for disease therapy, and provided valuable suggestions for further study in this field.

Bufalin engages in RIP1-dependent and ROS-dependent programmed necroptosis in breast cancer cells by targeting the RIP1/RIP3/PGAM5 pathway.

Breast cancer causes high mortality among females worldwide. Bufalin has recently been shown to trigger tumor cell death, although the mechanism of cytotoxicity remains unclear. The cytotoxicity of bufalin in breast cancer cells was examined using an MTT assay. The modes of death and intracellular reactive oxygen species production were measured by flow cytometry. We also observed cellular morphologic changes by Hoechst 33342 and propidium iodide staining and transmission electron microscopy. Western blotting was performed to determine the expression levels of related proteins. Our results showed that bufalin reduced cellular viability and promoted reactive oxygen species production, which could be inhibited by Nec-1 and N-acetylcysteine. Necroptosis was detected by Hoechst 33342 and propidium iodide staining and transmission electron microscopy. Western blot analysis showed that bufalin induced necroptosis by upregulating the necroptosis mediator RIP1 and the RIP1/RIP3/PGAM5 pathway. Taken together, these findings indicated that bufalin induces breast cancer cell necroptosis by targeting the RIP1/RIP3/PGAM5 pathway.

Nuclear pore protein TPR associates with lamin B1 and affects nuclear lamina organization and nuclear pore distribution.

The organization of the nuclear periphery is crucial for many nuclear functions. Nuclear lamins form dense network at the nuclear periphery and play a substantial role in chromatin organization, transcription regulation and in organization of nuclear pore complexes (NPCs). Here, we show that TPR, the protein located preferentially within the nuclear baskets of NPCs, associates with lamin B1. The depletion of TPR affects the organization of lamin B1 but not lamin A/C within the nuclear lamina as shown by stimulated emission depletion microscopy. Finally, reduction of TPR affects the distribution of NPCs within the nuclear envelope and the effect can be reversed by simultaneous knock-down of lamin A/C or the overexpression of lamin B1. Our work suggests a novel role for the TPR at the nuclear periphery: the TPR contributes to the organization of the nuclear lamina and in cooperation with lamins guards the interphase assembly of nuclear pore complexes.

RIP1 and RIP3 contribute to Tributyltin-induced toxicity in vitro and in vivo.

Tributyltin (TBT), a widely distributed environmental pollutant, is toxic to animals and human beings. Although its toxicity, especially the immunosuppressive effect, has been reported a lot, the underlying molecular mechanisms are still unclear. In this study, we investigated the mechanisms of TBT-induced cytotoxicity both in vitro and in vivo. TBT induced cell death in both J774A.1 macrophages and mouse bone marrow-derived macrophages (BMDMs) as measured by the LDH and Annexin V-FITC/PI dual staining assays. Pretreatment with RIP1 inhibitor Necrostatin-1 (Nec-1) or transfection with Rip1 siRNA significantly suppressed TBT-induced cytotoxicity in J774A.1 macrophages or human embryonic kidney cell line (HEK293 cells). TBT-induced cell death was also markedly inhibited in RIP3-/- BMDMs. In agreement with in vitro results, TBT-induced in vivo immunotoxic effects including leukocyte depletion and thymus atrophy were significantly attenuated in RIP3-/- mice or WT mice treated with Nec-1. Notably, the mortality rate induced by TBT was remarkably reduced in RIP3-/- mice (100% vs. 12.5% lethality) or Nec-1-treated mice (100% vs. 59.2% lethality) respectively. These results reveal a critical role of RIP1 and RIP3 in TBT-induced toxicity both in vitro and in vivo.

The specific RIP1 inhibitor necrostatin-1 ameliorated degradation of ECM in human SW1353 cells.

Abnormal destruction of the components of the articular extracellular matrix (ECM) such as type II collagen and aggrecan caused by advanced glycation end products (AGEs) has been considered as one of the pathological characteristics of osteoarthritis (OA). Receptor-interacting protein 1 (RIP1), an important serine/threonine kinase, possesses a variety of biological functions including cell proliferation, survival and death. The physiological roles of RIP1 in OA have not been reported before. Here, we found that AGEs increased the expression of RIP1 in human chondrosarcoma cell line SW1353 cells. Importantly, we found that antagonism of RIP1 using its specific inhibitor necrostatin-1 (Nec-1) ameliorated AGE-induced degradation of type II collagen and aggrecan in SW1353 cells. We also found that treatment with Nec-1 reduced the expression of MMP-3 and MMP-13 but restored the expression of Tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. Also, our results indicate that Nec-1 inhibited AGE-induced expression of ADAMTS-4 and ADAMTS-5. Mechanistically, we found that Nec-1 treatment inhibited the activation of JNK and the transcriptional factor AP-1 by reducing the expressions of c-Fos and c-Jun, the two main components of AP-1. Additionally, we found that Nec-1 treatment abolished AGE-induced activation of the transcriptional factor NF-κB by suppressing the nuclear translocation of p65. These findings suggest that RIP1 might be an important therapeutic target of OA.

RIP1 has a role in CD40-mediated apoptosis in human follicular lymphoma cells.

CD40 is a cell surface receptor which belongs to tumor necrosis factor receptor (TNFR) family members. It transmits signals that regulate diverse cellular responses such as proliferation, differentiation, adhesion molecule expression and apoptosis. Unlike other TNFR family members (TRAIL-R, Fas-R and TNFR1), the CD40 cytoplasmic tail lacks death domain. However, CD40 is capable of inducing apoptosis in different types of cancer cells including lymphoma. The apoptotic effect of CD40 is linked to the involvement of Fas, TRAIL or receptor interacting protein 1 (RIP1) kinase. We have previously shown that CD40 activation has anti-apoptotic or apoptotic effect in follicular lymphoma (FL) cell lines. In this study, we investigated the mechanism by which CD40 mediates apoptosis in a follicular lymphoma cell line, HF4.9. We show here that CD40-induced apoptosis was dependent on caspase-8 activation because caspase-8 specific inhibitor, Z-IETD-FMK completely prevented apoptosis. Therefore, the involvement of TRAIL, Fas and RIP1 in caspase-8 activation was examined. The exogenous TRAIL-induced apoptosis was fully prevented by anti-TRAIL neutralizing antibody. However, the antibody had no effect on CD40-induced apoptosis indicating that CD40 did not induce the expression of endogenous TRAIL in HF4.9 cells. Moreover, the cells were not sensitive to Fas-mediated apoptosis. Interestingly, RIP1 specific inhibitor, necrostatin-1 decreased CD40-induced apoptosis, which showed that RIP1 has a role in caspase-8 activation. In conclusion, the survival or apoptotic effects of CD40-mediated signaling might be related to the differentiation stages of FL cells.

Cytosolic calcium mediates RIP1/RIP3 complex-dependent necroptosis through JNK activation and mitochondrial ROS production in human colon cancer cells.

Necroptosis is a form of programmed necrosis mediated by signaling complexes with receptor-interacting protein 1 (RIP1) and RIP3 kinases as the main mediators. However, the underlying execution pathways of this phenomenon have yet to be elucidated in detail. In this study, a RIP1/RIP3 complex was formed in 2-methoxy-6-acetyl-7-methyljuglone (MAM)-treated HCT116 and HT29 colon cancer cells. With this formation, mitochondrial reactive oxygen species (ROS) levels increased, mitochondrial depolarization occurred, and ATP concentrations decreased. This process was identified as necroptosis. This finding was confirmed by experiments showing that MAM-induced cell death was attenuated by the pharmacological or genetic blockage of necroptosis signaling, including RIP1 inhibitor necrostatin-1s (Nec-1s) and siRNA-mediated gene silencing of RIP1 and RIP3, but was unaffected by caspase inhibitor z-vad-fmk or necrosis inhibitor 2-(1H-Indol-3-yl)-3-pentylamino-maleimide (IM54). Transmission electron microscopy (TEM) analysis further revealed the ultrastructural features of MAM-induced necroptosis. MAM-induced RIP1/RIP3 complex triggered necroptosis through cytosolic calcium (Ca2+) accumulation and sustained c-Jun N-terminal kinase (JNK) activation. Both calcium chelator BAPTA-AM and JNK inhibitor SP600125 could attenuate necroptotic features, including mitochondrial ROS elevation, mitochondrial depolarization, and ATP depletion. 2-thenoyltrifluoroacetone (TTFA), which is a mitochondrial complex II inhibitor, was found to effectively reverse both MAM induced mitochondrial ROS generation and cell death, indicating the complex II was the ROS-producing site. The essential role of mitochondrial ROS was confirmed by the protective effect of overexpression of manganese superoxide dismutase (MnSOD). MAM-induced necroptosis was independent of TNFα, p53, MLKL, and lysosomal membrane permeabilization. In summary, our study demonstrated that RIP1/RIP3 complex-triggered cytosolic calcium accumulation is a critical mediator in MAM-induced necroptosis through sustained JNK activation and mitochondrial ROS production. Our study also provided new insights into the molecular regulation of necroptosis in human colon cancer cells.

Molecular Pathways: The Necrosome-A Target for Cancer Therapy.

Necroptosis is a caspase-8-independent cell death that requires coactivation of receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3) kinases. The necrosome is a complex consisting of RIP1, RIP3, and Fas-associated protein with death domain leading to activation of the pseudokinase mixed lineage kinase like followed by a rapid plasma membrane rupture and inflammatory response through the release of damage-associated molecular patterns and cytokines. The necrosome has been shown to be relevant in multiple tumor types, including pancreatic adenocarcinoma, melanoma, and several hematologic malignancies. Preclinical data suggest that targeting this complex can have differential impact on tumor progression and that the effect of necroptosis on oncogenesis is cell-type and context dependent. The emerging data suggest that targeting the necrosome may lead to immunogenic reprogramming in the tumor microenvironment in multiple tumors and that combining therapies targeting the necrosome with either conventional chemotherapy or immunotherapy may have beneficial effects. Thus, understanding the interplay of necroptotic cell death, transformed cells, and the immune system may enable the development of novel therapeutic approaches. Clin Cancer Res; 23(5); 1132-6. ©2016 AACR.

Sensitizing acute myeloid leukemia cells to induced differentiation by inhibiting the RIP1/RIP3 pathway.

Tumor necrosis factor-α (TNF-α)-induced RIP1/RIP3 (receptor-interacting protein kinase 1/receptor-interacting protein kinase 3)-mediated necroptosis has been proposed as an alternative strategy for treating apoptosis-resistant leukemia. However, we found that most acute myeloid leukemia (AML) cells, especially M4 and M5 subtypes, produce TNF and show basal level activation of RIP1/RIP3/MLKL signaling, yet do not undergo necroptosis. TNF, through RIP1/RIP3 signaling, prevents degradation of SOCS1, a key negative regulator of interferon-γ (IFN-γ) signaling. Using both pharmacologic and genetic assays, we show here that inactivation of RIP1/RIP3 resulted in reduction of SOCS1 protein levels and partial differentiation of AML cells. AML cells with inactivated RIP1/RIP3 signaling show increased sensitivity to IFN-γ-induced differentiation. RIP1/RIP3 inactivation combined with IFN-γ treatment significantly attenuated the clonogenic capacity of both primary AML cells and AML cell lines. This combination treatment also compromised the leukemogenic ability of murine AML cells in vivo. Our studies suggest that inhibition of RIP1/RIP3-mediated necroptotic signaling might be a novel strategy for the treatment of AML when combined with other differentiation inducers.

HOXA repression is mediated by nucleoporin Nup93 assisted by its interactors Nup188 and Nup205.

BACKGROUND: The nuclear pore complex (NPC) mediates nuclear transport of RNA and proteins into and out of the nucleus. Certain nucleoporins have additional functions in chromatin organization and transcription regulation. Nup93 is a scaffold nucleoporin at the nuclear pore complex which is associated with human chromosomes 5, 7 and 16 and with the promoters of the HOXA gene as revealed by ChIP-on-chip studies using tiling microarrays for these chromosomes. However, the functional consequences of the association of Nup93 with HOXA is unknown. RESULTS: Here, we examined the association of Nup93 with the HOXA gene cluster and its consequences on HOXA gene expression in diploid colorectal cancer cells (DLD1). Nup93 showed a specific enrichment ~1 Kb upstream of the transcription start site of each of the HOXA1, HOXA3 and HOXA5 promoters, respectively. Furthermore, the association of Nup93 with HOXA was assisted by its interacting partners Nup188 and Nup205. The depletion of the Nup93 sub-complex significantly upregulated HOXA gene expression levels. However, expression levels of a control gene locus (GLCCI1) on human chromosome 7 were unaffected. Three-dimensional fluorescence in situ hybridization (3D-FISH) analyses revealed that the depletion of the Nup93 sub-complex (but not Nup98) disengages the HOXA gene locus from the nuclear periphery, suggesting a potential role for Nup93 in tethering and repressing the HOXA gene cluster. Consistently, Nup93 knockdown increased active histone marks (H3K9ac), decreased repressive histone marks (H3K27me3) on the HOXA1 promoter and increased transcription elongation marks (H3K36me3) within the HOXA1 gene. Moreover, the combined depletion of Nup93 and CTCF (a known organizer of HOXA gene cluster) but not Nup93 alone, significantly increased GLCCI1 gene expression levels. Taken together, this suggests a novel role for Nup93 and its interactors in repressing the HOXA gene cluster. CONCLUSIONS: This study reveals that the nucleoporin Nup93 assisted by its interactors Nup188 and Nup205 mediates the repression of HOXA gene expression.

Synergistic effects of selective inhibitors targeting the PI3K/AKT/mTOR pathway or NUP214-ABL1 fusion protein in human Acute Lymphoblastic Leukemia.

Philadelphia chromosome-positive (Ph+) Acute Lymphoblastic Leukemia (ALL) accounts for 25-30% of adult ALL and its incidence increases with age in adults >40 years old. Irrespective of age, the ABL1 fusion genes are markers of poor prognosis and amplification of the NUP214-ABL1 oncogene can be detected mainly in patients with T-ALL. T cell malignancies harboring the ABL1 fusion genes are sensitive to many cytotoxic agents, but up to date complete remissions have not been achieved. The PI3K/Akt/mTOR signaling pathway is often activated in leukemias and plays a crucial role in leukemogenesis.We analyzed the effects of three BCR-ABL1 tyrosine kinase inhibitors (TKIs), alone and in combination with a panel of selective PI3K/Akt/mTOR inhibitors, on three NUP214-ABL1 positive T-ALL cell lines that also displayed PI3K/Akt/mTOR activation. Cells were sensitive to anti BCR-ABL1 TKIs Imatinib, Nilotinib and GZD824, that specifically targeted the ABL1 fusion protein, but not the PI3K/Akt/mTOR axis. Four drugs against the PI3K/Akt/mTOR cascade, GSK690693, NVP-BGT226, ZSTK474 and Torin-2, showed marked cytotoxic effects on T-leukemic cells, without affecting the NUP214-ABL1 kinase and related pathway. Dephosphorylation of pAkt and pS6 showed the cytotoxicity of these compounds. Either single or combined administration of drugs against the different targets displayed inhibition of cellular viability associated with a concentration-dependent induction of apoptosis, cell cycle arrest in G0/G1 phase and autophagy, having the combined treatments a significant synergistic cytotoxic effect. Co-targeting NUP214-ABL1 fusion gene and PI3K/Akt/mTOR signaling pathway could represent a new and effective pharmacological strategy to improve the outcome in NUP214-ABL1 positive T-ALL.

Identification of replication-dependent and replication-independent linker histone complexes: Tpr specifically promotes replication-dependent linker histone stability.

BACKGROUND: There are 11 variants of linker histone H1 in mammalian cells. Beyond their shared abilities to stabilize and condense chromatin, the H1 variants have been found to have non-redundant functions, the mechanisms of which are not fully understood. Like core histones, there are both replication-dependent and replication-independent linker histone variants. The histone chaperones and other factors that regulate linker histone dynamics in the cell are largely unknown. In particular, it is not known whether replication-dependent and replication-independent linker histones interact with distinct or common sets of proteins. To better understand linker histone dynamics and assembly, we used chromatography and mass spectrometry approaches to identify proteins that are associated with replication-dependent and replication-independent H1 variants. We then used a variety of in vivo analyses to validate the functional relevance of identified interactions. RESULTS: We identified proteins that bind to all linker histone variants and proteins that are specific for only one class of variant. The factors identified include histone chaperones, transcriptional regulators, RNA binding proteins and ribosomal proteins. The nuclear pore complex protein Tpr, which was found to associate with only replication-dependent linker histones, specifically promoted their stability. CONCLUSION: Replication-dependent and replication-independent linker histone variants can interact with both common and distinct sets of proteins. Some of these factors are likely to function as histone chaperones while others may suggest novel links between linker histones and RNA metabolism. The nuclear pore complex protein Tpr specifically interacts with histone H1.1 and H1.2 but not H1x and can regulate the stability of these replication-dependent linker histones.

Differential role of RIP1 in Smac mimetic-mediated chemosensitization of neuroblastoma cells.

We explored the potential of Smac mimetics, which antagonize Inhibitor of Apoptosis (IAP) proteins, for chemosensitization of neuroblastoma (NB). Here, we report that Smac mimetics, e.g. BV6, prime NB cells for chemotherapeutics including the topoisomerase II inhibitor doxorubicin (DOX) and vinca alkaloids such as Vincristine (VCR), Vinblastine (VBL) and Vinorelbine (VNR). Additionally, BV6 acts in concert with DOX or VCR to suppress long-term clonogenic growth. While BV6 causes rapid downregulation of cellular IAP (cIAP)1 protein and nuclear factor-kappaB (NF-κB) activation, DOX/BV6- or VCR/BV6-induced apoptosis occurs independently of NF-κB or TNFα signaling, since overexpression of dominant-negative IκBα superrepressor or the Tumor Necrosis Factor (TNF)α-blocking antibody Enbrel fail to block cell death. Mechanistic studies reveal that Receptor-interacting protein (RIP)1 is required for DOX/BV6-, but not for VCR/BV6-induced apoptosis, since transient or stable knockdown of RIP1 or the pharmacological RIP1 inhibitor necrostatin-1 significantly reduce apoptosis. By comparison, VCR/BV6-mediated apoptosis critically depends on the mitochondrial pathway. VCR/BV6 cotreatment causes phosphorylation of BCL-2 during mitotic arrest, enhanced activation of BAX and BAK and loss of mitochondrial membrane potential (MMP). Additionally, overexpression of BCL-2 profoundly suppresses VCR/BV6-induced apoptosis. Thus, BV6 sensitizes NB cells to chemotherapy-induced apoptosis via distinct initial signaling mechanisms depending on the chemotherapeutic drug. These findings provide novel mechanistic insights into Smac mimetic-mediated chemosensitization of NB.

CD40 ligand induces RIP1-dependent, necroptosis-like cell death in low-grade serous but not serous borderline ovarian tumor cells.

Ovarian high-grade serous carcinomas (HGSCs) and invasive low-grade serous carcinomas (LGSCs) are considered to be distinct entities. In particular, LGSCs are thought to arise from non-invasive serous borderline ovarian tumors (SBOTs) and show poor responsiveness to conventional chemotherapy. The pro-apoptotic effects of CD40 ligand (CD40L) have been demonstrated in HGSC, though the underlying mechanisms are not fully understood. Conversely, the therapeutic potential of the CD40L-CD40 system has yet to be evaluated in LGSC. We now show that CD40 protein is focally expressed on tumor cells in two of five primary LGSCs compared with no expression in eight primary SBOTs. Treatment with CD40L or agonistic CD40 antibody decreased the viability of LGSC-derived MPSC1 and VOA1312 cells, but not SBOT3.1 cells. Small interfering RNA (siRNA) targeting CD40 was used to show that it is required for these reductions in cell viability. CD40L treatment increased cleaved caspase-3 levels in MPSC1 cells though, surprisingly, neither pan-caspase inhibitor nor caspase-3 siRNA reversed or even attenuated CD40L-induced cell death. In addition, CD40-induced cell death was not affected by knockdown of the mitochondrial proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG). Interestingly, CD40L-induced cell death was blocked by necrostatin-1, an inhibitor of receptor-interacting protein 1 (RIP1), and attenuated by inhibitors of RIP3 (GSK'872) or MLKL (mixed lineage kinase domain-like; necrosulfonamide). Our results indicate that the upregulation of CD40 may be relatively common in LGSC and that CD40 activation induces RIP1-dependent, necroptosis-like cell death in LGSC cells.

Protease 3C of hepatitis A virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death.

BACKGROUND: 3C proteases, the main proteases of picornaviruses, play the key role in viral life cycle by processing polyproteins. In addition, 3C proteases digest certain host cell proteins to suppress antiviral defense, transcription, and translation. The activity of 3C proteases per se induces host cell death, which makes them critical factors of viral cytotoxicity. To date, cytotoxic effects have been studied for several 3C proteases, all of which induce apoptosis. This study for the first time describes the cytotoxic effect of 3C protease of human hepatitis A virus (3Cpro), the only proteolytic enzyme of the virus. RESULTS: Individual expression of 3Cpro induced catalytic activity-dependent cell death, which was not abrogated by the pan-caspase inhibitor (z-VAD-fmk) and was not accompanied by phosphatidylserine externalization in contrast to other picornaviral 3C proteases. The cell survival was also not affected by the inhibitors of cysteine proteases (z-FA-fmk) and RIP1 kinase (necrostatin-1), critical enzymes involved in non-apoptotic cell death. A substantial fraction of dying cells demonstrated numerous non-acidic cytoplasmic vacuoles with not previously described features and originating from several types of endosomal/lysosomal organelles. The lysosomal protein Lamp1 and GTPases Rab5, Rab7, Rab9, and Rab11 were associated with the vacuolar membranes. The vacuolization was completely blocked by the vacuolar ATPase inhibitor (bafilomycin A1) and did not depend on the activity of the principal factors of endosomal transport, GTPases Rab5 and Rab7, as well as on autophagy and macropinocytosis. CONCLUSIONS: 3Cpro, apart from other picornaviral 3C proteases, induces caspase-independent cell death, accompanying by cytoplasmic vacuolization. 3Cpro-induced vacuoles have unique properties and are formed from several organelle types of the endosomal/lysosomal compartment. The data obtained demonstrate previously undocumented morphological characters of the 3Cpro-induced cell death, which can reflect unknown aspects of the human hepatitis A virus-host cell interaction.